Method for treating diseases associated with oxidative stress or endothelial dysfunction using substituted 1-pyrrolidinyloxy, 1-piperidinyloxy and 1-azepanyloxy compounds

ABSTRACT

A method reduces oxidative stress or endothelial dysfunction in a mammal. The method includes administering to the mammal a compound of formula I: 
                         
or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt of the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

Any and all priority claims identified in the application data sheet, orany correction thereto, are hereby incorporated by reference under 37C.F.R. 1.57 for example, this application is a divisional of and claimspriority to U.S. application Ser. No. 14/769,078, filed Aug. 19, 2015which is the U.S. National Phase of International Application No.PCT/IB2014/059451 filed Mar. 5, 2014, designating the U.S. and publishedas WO 2014/136059 on Sep. 12, 2014 which claims the benefit of U.S.Provisional Application No. 61/772,861 filed Mar. 5, 2013, each of whichis incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention provides prodrugs of compounds comprising a nitricoxide donor and a reactive oxygen species (ROS) degradation catalyst,and pharmaceutical compositions thereof.

BACKGROUND ART

U.S. Pat. Nos. 6,448,267, 6,455,542 and 6,759,430 disclose, inter alia,1-pyrrolidinyloxy, 1-piperidinyloxy and 1-azepanyloxy derivativescomprising a nitric oxide donor and a O₂ ⁻ scavenger, capable of actingas sources of nitric oxide and as ROS degradation catalysts, theirpreparation, and their use in the treatment of various conditionsassociated with oxidative stress or endothelial dysfunction such asdiabetes mellitus and cardiovascular diseases.

International Publication No. WO 2012/093383 discloses methods andcompositions for treatment of sepsis and conditions associated therewithusing the compounds disclosed in U.S. Pat. Nos. 6,448,267, 6,455,542 and6,759,430; International Publication No. WO 2011/092690 disclosesmethods and compositions for prevention, treatment, or management ofpulmonary arterial hypertension (PAH) using those compounds;International Publication No. WO 2013/005216 discloses methods andcompositions for prevention and treatment of renal ischemia-reperfusioninjury; and International Publication No. WO 2013/190497 disclosesmethods and compositions for treatment of Cl₂ inhalational lung injury(CILI). The entire contents of each and all these patents and patentpublications being herewith incorporated by reference in their entiretyas if fully disclosed herein.

As shown in International Publication No. WO 2013/005216, the compoundsdisclosed in U.S. Pat. Nos. 6,448,267, 6,455,542 and 6,759,430, inparticular 3-nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy, hereinidentified R-100, that is specifically exemplified in the aforesaidInternational publications for treatment of sepsis, PAH, renalischemia-reperfusion injury, and CILI, are highly insoluble in water butare soluble in certain organic solvents such as dimethyl sulfoxide(DMSO) or alternatively, when formulated as inclusion complexes withhydroxyalkylcyclodextrin such as hydroxypropylcyclodextrin (HPCD). Theinclusion of such organic solvents in pharmaceutical compositions ispotentially toxic and is thus preferably avoided. HPCD is relativelywell tolerated but must be admixed with R-100 in a ratio of >20:1(HPCD:R-100) in order to fully dissolve said compound. The upper limitof safety for clinical administration of HPCD is unknown, but thegreatest amount approved for use in humans via an intravenous route is adaily quantity of 7 grams. Given the minimum usable ratio of 20:1 forthe dissolution of R-100 and HPCD, and the limit of 7 grams of HPCD perday, the maximal amount of R-100 delivered in such an intravenousformulation would be 350 mg daily.

SUMMARY OF INVENTION

In order to overcome the limitations presented above, prodrugs of R-100,e.g., 2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-yl acetate,herein identified R-107, have been prepared. As surprisingly found,R-107 is stable oil until exposure to plasma, after which it is readilyconverted to its corresponding 1-pyrrolidinyloxy derivative R-100. R-107has a density of 1.12 mg/ml and may be administered in either pure formor diluted, e.g., in polyethyleneglycol (PEG)-400, via diverse routesincluding intravenous, intramuscular, subcutaneous, and topical, e.g.,to the skin, wounds, ulcers, oral cavity, vagina, and anal canal.

In one aspect, the present invention thus provides a compound of thegeneral formula I:

or an enantiomer, diastereomer, racemate, or pharmaceutically acceptablesalt or solvate thereof,

wherein

R₁ each independently is selected from H, —OH, —COR₄, —COOR₄, —OCOOR₄,—OCON(R₄)₂, —(C₁-C₁₆)alkylene-COOR₄, —CN, —NO₂, —SH, —SR₄,—(C₁-C₁₆)alkyl, —O—(C₁-C₁₆)alkyl, —N(R₄)₂, —CON(R₄)₂, —SO₂R₄, —SO₂NHR₄,—S(═O)R₄, or a nitric oxide donor group of the formula —X₁—X₂—X₃,wherein X₁ is absent or selected from —O—, —S— or —NH—; X₂ is absent oris (C₁-C₂₀)alkylene optionally substituted by one or more —ONO₂ groups;and X₃ is —NO or —ONO₂, provided that at least one R₁ group is a nitricoxide donor group;

R₂ each independently is selected from (C₁-C₁₆)alkyl, (C₂-C₁₆)alkenyl,or (C₂-C₁₆)alkynyl;

R₃ is selected from (C₁-C₁₀)alkyl, (C₃-C₁₀)cycloalkyl, (C₆-C₁₄)aryl, or4-12-membered heterocyclyl, each of which may optionally be substitutedwith —OH, —COR₅, —COOR₅, —(C₁-C₈)alkylene-COOR₅, —CN, —NO₂,—(C₁-C₈)alkyl, —O—(C₁-C₈)alkyl, —N(R₅)₂, —CON(R₅)₂, —SO₂R₅, —SO₂NHR₅, or—S(═O)R₅;

R₄ each independently is selected from H, (C₁-C₈)alkyl,(C₃-C₁₀)cycloalkyl, (C₆-C₁₄)aryl, or 4-12-membered heterocyclyl, each ofwhich other than H may optionally be substituted with —OH, —COR₅,—COOR₅, —OCOOR₅, —OCON(R₅)₂, —(C₁-C₈)alkylene-COOR₅, —CN, —NO₂, —SH,—SR₅, —(C₁-C₈)alkyl, —O—(C₁-C₈)alkyl, —N(R₅)₂, —CON(R₅)₂, —SO₂R₅,—SO₂NHR₅, or —S(═O)R₅;

R₅ each independently is selected from H, (C₁-C₈)alkyl,(C₃-C₁₀)cycloalkyl, (C₆-C₁₄)aryl, or 4-12-membered heterocyclyl; and

n is an integer of 1 to 3.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of the general formula I as definedabove, or an enantiomer, diastereomer, racemate, or pharmaceuticallyacceptable salt or solvate thereof, and a pharmaceutically acceptablecarrier. The compounds and pharmaceutical compositions of the presentinvention are useful for prevention, treatment or management of adisease, disorder or condition associated with oxidative stress orendothelial dysfunction.

In a further aspect, the present invention relates to a compound of thegeneral formula I as defined above, or an enantiomer, diastereomer,racemate, or pharmaceutically acceptable salt or solvate thereof, foruse in prevention, treatment or management of a disease, disorder orcondition associated with oxidative stress or endothelial dysfunction.

In yet another aspect, the present invention relates to use of acompound of the general formula I as defined above, or an enantiomer,diastereomer, racemate, or pharmaceutically acceptable salt or solvatethereof, for the preparation of a pharmaceutical composition forprevention, treatment or management of a disease, disorder or conditionassociated with oxidative stress or endothelial dysfunction.

In still another aspect, the present invention relates to a method forprevention, treatment or management of a disease, disorder or conditionassociated with oxidative stress or endothelial dysfunction in anindividual in need thereof, comprising administering to said individualan effective amount of a compound of the general formula I as definedabove, or an enantiomer, diastereomer, racemate, or pharmaceuticallyacceptable salt or solvate thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1F show representative photomicrographs demonstratinghematoxylin/eosin-stained lung sections taken from sham-operated mice(1A, 1B); animals treated with Cl₂+HPCD (1C); animals treated withCl₂+saline (1D); animals treated with Cl₂+R-100, 80 mg/kg/dose, IP (1E);and animals treated with Cl₂+R-107, 100 mg/kg/dose, IP (1F), asdescribed in Example 16.

FIG. 2 shows that R-107 (100 mg/kg/dose) as well as its corresponding1-pyrrolidinyloxy, R-100 (80 mg/kg/dose), when administered 2 and 6hours post a 60 minute exposure to Cl₂ (400 ppm) containing air,significantly attenuated CILI in mice 24 hours post exposure asexemplified by the improved histology scores.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides 1-pyrrolidinyl-,1-piperidinyl- and 1-azepanyl-ester derivatives of the general formula Ias defined above, comprising one to four nitric oxide donor groups and aROS degradation catalyst, i.e., a superoxide anion (O₂ ⁻) scavenger. Thecompounds of the present invention are, in fact, prodrugs for thecorresponding hydroxylamine or N-hydroxyl (N—OH) compounds uponhydrolysis of the ester bond (O—C(O)R₃), and those hydroxylaminecompounds are then oxidized, in vivo, to their corresponding nitroxidederivatives, more particularly 1-pyrrolidinyloxy, 1-piperidinyloxy, and1-azepanyloxy derivatives disclosed in the aforesaid U.S. Pat. Nos.6,448,267, 6,455,542 and 6,759,430. The compounds of the presentinvention are thus expected to be effective in all those clinicalindications wherein the aforesaid 1-pyrrolidinyloxy, 1-piperidinyloxy,and 1-azepanyloxy derivatives are of benefit.

The term “alkyl” as used herein typically means a straight or branchedsaturated hydrocarbon radical having 1-16 carbon atoms and includes,e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, isoamyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl,n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl,n-tetradecyl, n-pentadecyl, n-hexadecyl, and the like. Preferred are(C₁-C₈)alkyl groups, more preferably (C₁-C₄)alkyl groups, mostpreferably methyl and ethyl. The terms “alkenyl” and “alkynyl” typicallymean straight and branched hydrocarbon radicals having 2-16 carbon atomsand 1 double or triple bond, respectively, and include ethenyl,propenyl, 3-buten-1-yl, 2-ethenylbutyl, 3-octen-1-yl, 3-nonenyl,3-decenyl, and the like, and propynyl, 2-butyn-1-yl, 3-pentyn-1-yl,3-hexynyl, 3-octynyl, 4-decynyl, and the like. C₂-C₆ alkenyl and alkynylradicals are preferred, more preferably C₂-C₄ alkenyl and alkynyl.

The term “alkylene” typically means a divalent straight or branchedhydrocarbon radical having 1-20 carbon atoms and includes, e.g.,methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene,2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene,2,3-dimethylbutylene, heptylene, octylene, and the like. Preferred are(C₁-C₈)alkylene, more preferably (C₁-C₄)alkylene, most preferably(C₁-C₂)alkylene.

The term “cycloalkyl” as used herein means a cyclic or bicyclichydrocarbyl group having 3-10 carbon atoms such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl,bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like. Preferred are(C₅-C₁₀)cycloalkyls, more preferably (C₅-C₇)cycloalkyls.

The term “aryl” denotes an aromatic carbocyclic group having 6-14 carbonatoms consisting of a single ring or multiple rings either condensed orlinked by a covalent bond such as, but not limited to, phenyl, naphthyl,phenanthryl, and biphenyl.

The term “heterocyclic ring” denotes a mono- or poly-cyclic non-aromaticring of 4-12 atoms containing at least one carbon atom and one to threeheteroatoms selected from sulfur, oxygen or nitrogen, which may besaturated or unsaturated, i.e., containing at least one unsaturatedbond. Preferred are 5- or 6-membered heterocyclic rings. The term“heterocyclyl” as used herein refers to any univalent radical derivedfrom a heterocyclic ring as defined herein by removal of hydrogen fromany ring atom. Examples of such radicals include, without limitation,piperidino, 4-morpholinyl, or pyrrolidinyl.

The term “nitric oxide donor group” as defined herein refers to anygroup of the formula —X₁—X₂—X₃, wherein X₁ may be absent or is selectedfrom —O—, —S— or —NH—; X₂ may be absent or is (C₁-C₂₀)alkyleneoptionally substituted by one or more —ONO₂ groups; and X₃ is —NO or—ONO₂. Particular nitric oxide donor groups are those in which X₁ isabsent or is —O—; X₂ is absent or is —(C₁-C₆)alkylene, preferably—(C₁-C₃)alkylene, more preferably methylene; X₃ is —NO or —ONO₂,preferably —ONO₂, and said alkylene is optionally substituted as definedhereinabove. According to the invention, the compound of the generalformula I may comprise one nitric oxide donor group or more than oneidentical or different nitric oxide donor groups.

In certain embodiments, the compound of the present invention is acompound of the general formula I, wherein R₁ each independently is H,—COOR₄, —CON(R₄)₂, or a nitric oxide donor group; and R₄ is H.

In certain embodiments, the compound of the present invention is acompound of the general formula I, wherein R₂ each independently is(C₁-C₈)alkyl, preferably (C₁-C₄)alkyl, more preferably methyl or ethyl.In particular such embodiments, the R₂ groups are identical.

In certain embodiments, the compound of the present invention is acompound of the general formula I, wherein R₃ is (C₁-C₈)alkyl,preferably (C₁-C₃)alkyl, optionally substituted, preferably at aterminal carbon atom, with —OH, —N(R₅)₂, or —COOR₅, wherein R₅ eachindependently is (C₁-C₈)alkyl, preferably (C₁-C₂)alkyl, or H. Inparticular such embodiments, R₃ is (C₁-C₈)alkyl, preferably(C₁-C₃)alkyl, (C₁-C₈)alkylene-OH, preferably (C₁-C₃)alkylene-OH,(C₁-C₈)alkylene-N(R₅)₂, preferably (C₁-C₃)alkylene-N(R₅)₂, or(C₁-C₈)alkylene-COOR₅, preferably (C₁-C₃)alkylene-COOR₅.

In certain embodiments, the compound of the present invention is acompound of the general formula I, wherein in said nitric oxide donorgroup, X₁ is absent or —O—; X₂ is absent or (C₁-C₂₀)alkylene, preferably—(C₁-C₆)alkylene, more preferably —(C₁-C₃)alkylene, most preferablymethylene; X₃ is —NO or —ONO₂, preferably —ONO₂; and said alkylene isoptionally substituted by one or more —ONO₂ groups.

In certain embodiments, the compound of the present invention is acompound of the general formula I, wherein R₁ each independently is H,—COOR₄, —CON(R₄)₂, or a nitric oxide donor group; R₂ each independentlyis (C₁-C₈)alkyl, preferably (C₁-C₄)alkyl, more preferably methyl orethyl; R₃ is (C₁-C₈)alkyl, preferably (C₁-C₃)alkyl, optionallysubstituted, preferably at a terminal carbon atom, with —OH, —N(R₅)₂, or—COOR₅; R₄ is H; R₅ each independently is (C₁-C₈)alkyl, preferably(C₁-C₂)alkyl, or H; and in said nitric oxide donor group, X₁ is absentor —O—; X₂ is absent or (C₁-C₂₀)alkylene, preferably —(C₁-C₆)alkylene,more preferably —(C₁-C₃)alkylene, most preferably methylene; X₃ is —NOor —ONO₂, preferably —ONO₂; and said alkylene is optionally substitutedby one or more —ONO₂ groups.

In certain embodiments, the compound of the present invention is acompound of the general formula I, wherein R₁ to R₅ and the nitric oxidedonor group(s) each independently is selected from the options definedby any one of the embodiments above; and n is 1, 2 or 3, i.e., a1-pyrrolidinyl ester derivative of the formula Ia, 1-piperidinyl esterderivative of the formula Ib, or 1-azepanyl ester derivative of theformula Ic (see Table 1).

In certain particular embodiments, the compound of the present inventionis a compound of the formula Ia in Table 1, wherein either the carbonatom at position 3 of the pyrrolidine ring or the carbon atom atposition 4 of the pyrrolidine ring, or both, are each linked to a nitricoxide donor group. More particular such embodiments are those whereineach one of the nitric oxide donor groups independently is of theformula —(C₁-C₆)alkylene-ONO₂, preferably —(C₁-C₃)alkylene-ONO₂, morepreferably —CH₂—ONO₂, or —O—(C₁-C₆)alkylene-ONO₂, wherein said alkyleneis optionally substituted by one or more —ONO₂ groups, or is —ONO₂.

In other particular embodiments, the compound of the present inventionis a compound of the formula Ib in Table 1, wherein one, two or three ofthe carbon atoms at positions 3 to 5 of the piperidine ring are eachlinked to a nitric oxide donor group, i.e., (i) the carbon atom atposition 3 of the piperidine ring and optionally one or more of thecarbon atoms at positions 4 or 5 of the piperidine ring are each linkedto a nitric oxide donor group; (ii) the carbon atom at position 4 of thepiperidine ring and optionally one or more of the carbon atoms atpositions 3 or 5 of the piperidine ring are each linked to a nitricoxide donor group; or (iii) the carbon atom at position 5 of thepiperidine ring and optionally one or more of the carbon atoms atpositions 3 or 4 of the piperidine ring are each linked to a nitricoxide donor group. More particular such embodiments are those whereineach one of the nitric oxide donor groups independently is of theformula —(C₁-C₆)alkylene-ONO₂, preferably —(C₁-C₃)alkylene-ONO₂, morepreferably —CH₂—ONO₂, or —O—(C₁-C₆)alkylene-ONO₂, wherein said alkyleneis optionally substituted by one or more —ONO₂ groups, or is —ONO₂.

In further particular embodiments, the compound of the present inventionis a compound of the formula Ic in Table 1, wherein one, two, three orfour of the carbon atoms at positions 3 to 6 of the azepane ring areeach linked to a nitric oxide donor group, i.e., (i) the carbon atom atposition 3 of the azepane ring and optionally one or more of the carbonatoms at positions 4 to 6 of the azepane ring are each linked to anitric oxide donor group; (ii) the carbon atom at position 4 of theazepane ring and optionally one or more of the carbon atoms at positions3, 5 or 6 of the azepane ring are each linked to a nitric oxide donorgroup; (iii) the carbon atom at position 5 of the azepane ring andoptionally one or more of the carbon atoms at positions 3, 4 or 6 of theazepane ring are each linked to a nitric oxide donor group; or (iv) thecarbon atom at position 6 of the azepane ring and optionally one or moreof the carbon atoms at positions 3 to 5 of the azepane ring are eachlinked to a nitric oxide donor group. More particular such embodimentsare those wherein each one of the nitric oxide donor groupsindependently is of the formula —(C₁-C₆)alkylene-ONO₂, preferably—(C₁-C₃)alkylene-ONO₂, more preferably —CH₂—ONO₂, or—O—(C₁-C₆)alkylene-ONO₂, wherein said alkylene is optionally substitutedby one or more —ONO₂ groups, or is —ONO₂.

TABLE 1 Structures Ia, Ib, and Ic, indicating 1-pyrrolidinyl-,1-piperidinyl- and 1-azepanyl-ester derivatives, respectively

Ia

Ib

Ic

Specific compounds of the general formulas Ia, Ib, and Ic describedherein, in which each one of the R₁ groups independently is either H orthe nitric oxide donor group —CH₂—ONO₂, and R₃ is methyl, ethyl, orisopropyl, are herein identified compounds 1a₁₋₃ to 15a₁₋₃ in bold(compound 1a₁ is also identified R-107), and their full chemicalstructures are depicted in Table 2. Similar compounds in which each oneof the nitric oxide donor groups is —ONO₂ rather than —CH₂—ONO₂ areherein identified compounds 1b₁₋₃ to 15b₁₋₃ in bold. Other specificcompounds of the general formulas Ia and Ib described herein, in whichone R₁ group is the nitric oxide donor group —CH₂—ONO₂, another R₁ groupis not H, and R₃ is methyl, ethyl, or isopropyl, are herein identifiedcompounds 16a₁₋₃ and 17a₁₋₃ in bold, and their full chemical structuresare depicted in Table 3. Similar compounds in which the nitric oxidedonor group is —ONO₂ rather than —CH₂—ONO₂ are herein identifiedcompounds 16b₁₋₃ and 17b₁₋₃ in bold. Further specific compounds of thegeneral formula Ib described herein, in which one R₁ group is the nitricoxide donor group —O—CH₂—CH(ONO₂)CH₂—ONO₂, the other R₁ groups are H,and R₃ is methyl, ethyl, or isopropyl, are herein identified compounds18₁₋₃ in bold, and their full chemical structure is depicted in Table 3.

In specific embodiments, the compound of the invention is the compoundof formula Ia, i.e., a compound of the general formula I in which n is1, wherein R₂ each is methyl; R₃ is methyl, ethyl, or isopropyl; and (i)the R₁ group linked to the carbon atom at position 3 of the pyrrolidinering is the nitric oxide donor group —CH₂—ONO₂; and the R₁ group linkedto the carbon atom at position 4 of the pyrrolidine ring is H, i.e.,2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-yl acetate,2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-yl propionate, and2,2,5,5-tetramethyl-3-(nitrooxymethyl) pyrrolidin-1-yl isobutyrate(compounds 1a₁, 1a₂ and 1a₃, respectively); (i) the R₁ group linked tothe carbon atom at position 3 of the pyrrolidine ring is the nitricoxide donor group —ONO₂; and the R₁ group linked to the carbon atom atposition 4 of the pyrrolidine ring is H, i.e.,2,2,5,5-tetramethyl-3-(nitrooxy)pyrrolidin-1-yl acetate,2,2,5,5-tetramethyl-3-(nitrooxy) pyrrolidin-1-yl propionate, and2,2,5,5-tetramethyl-3-(nitrooxy)pyrrolidin-1-yl isobutyrate (hereinidentified compounds 1b₁, 1b₂ and 1b₃, respectively); (iii) each one ofthe R₁ groups linked to the carbon atoms at positions 3 and 4 of thepyrrolidine ring is the nitric oxide donor group —CH₂—ONO₂, i.e.,2,2,5,5-tetramethyl-3,4-bis(nitrooxymethyl) pyrrolidin-1-yl acetate,2,2,5,5-tetramethyl-3,4-bis(nitrooxymethyl) pyrrolidin-1-yl propionate,and 2,2,5,5-tetramethyl-3,4-bis(nitrooxymethyl)pyrrolidin-1-ylisobutyrate (herein identified compounds 2a₁, 2a₂ and 2a₃,respectively); or (iv) each one of the R₁ groups linked to the carbonatoms at positions 3 and 4 of the pyrrolidine ring is the nitric oxidedonor group —ONO₂, i.e.,2,2,5,5-tetramethyl-3,4-bis(nitrooxy)pyrrolidin-1-yl acetate,2,2,5,5-tetramethyl-3,4-bis(nitrooxy) pyrrolidin-1-yl propionate, and2,2,5,5-tetramethyl-3,4-bis(nitrooxy)pyrrolidin-1-yl isobutyrate (hereinidentified compounds 2b₁, 2b₂ and 2_(b3), respectively).

In other specific embodiments, the compound of the invention is thecompound of formula Ib, i.e., a compound of the general formula Iwherein n is 2, wherein R₂ each is methyl; R₃ is methyl, ethyl, orisopropyl; and (i) the R₁ group linked to the carbon atom at position 3of the piperidine ring is the nitric oxide donor group —CH₂—ONO₂; andeach one of the R₁ groups linked to the carbon atoms at positions 4 and5 of the piperidine ring is H, i.e.,2,2,6,6-tetramethyl-3-(nitrooxymethyl)piperidin-1-yl acetate,2,2,6,6-tetramethyl-3-(nitrooxymethyl) piperidin-1-yl propionate, and2,2,6,6-tetramethyl-3-(nitrooxymethyl) piperidin-1-yl isobutyrate(herein identified compounds 3a₁, 3a₂ and 3a₃, respectively); (ii) theR₁ group linked to the carbon atom at position 3 of the piperidine ringis the nitric oxide donor group —ONO₂; and each one of the R₁ groupslinked to the carbon atoms at positions 4 and 5 of the piperidine ringis H, i.e., 2,2,6,6-tetramethyl-3-(nitrooxy)piperidin-1-yl acetate,2,2,6,6-tetramethyl-3-(nitrooxy)piperidin-1-yl propionate, and2,2,6,6-tetramethyl-3-(nitrooxy)piperidin-1-yl isobutyrate (hereinidentified compounds 3b₁, 3b₂ and 3b₃, respectively); (iii) the R₁ grouplinked to the carbon atom at position 4 of the piperidine ring is thenitric oxide donor group —CH₂—ONO₂; and each one of the R₁ groups linkedto the carbon atoms at positions 3 and 5 of the piperidine ring is H,i.e., 2,2,6,6-tetramethyl-4-(nitrooxymethyl) piperidin-1-yl acetate,2,2,6,6-tetramethyl-4-(nitrooxymethyl)piperidin-1-yl propionate, and2,2,6,6-tetramethyl-4-(nitrooxymethyl) piperidin-1-yl isobutyrate(herein identified compounds 4a₁, 4a₂ and 4a₃, respectively); (iv) theR₁ group linked to the carbon atom at position 4 of the piperidine ringis the nitric oxide donor group —ONO₂; and each one of the R₁ groupslinked to the carbon atoms at positions 3 and 5 of the piperidine ringis H, i.e., 2,2,6,6-tetramethyl-4-(nitrooxy)piperidin-1-yl acetate,2,2,6,6-tetramethyl-4-(nitrooxy)piperidin-1-yl propionate, and2,2,6,6-tetramethyl-4-(nitrooxy)piperidin-1-yl isobutyrate (hereinidentified compounds 4b₁, 4b₂ and 4b₃, respectively); (v) each one ofthe R₁ groups linked to the carbon atoms at positions 3 and 4 of thepiperidine ring is the nitric oxide donor group —CH₂—ONO₂; and the R₁group linked to the carbon atom at position 5 of the piperidine ring isH, i.e., 2,2,6,6-tetramethyl-3,4-bis(nitrooxymethyl)piperidin-1-ylacetate, 2,2,6,6-tetramethyl-3,4-bis(nitrooxymethyl)piperidin-1-ylpropionate, and2,2,6,6-tetramethyl-3,4-bis(nitrooxymethyl)piperidin-1-yl isobutyrate(herein identified compounds 5a₁, 5a₂ and 5a₃, respectively); (vi) eachone of the R₁ groups linked to the carbon atoms at positions 3 and 4 ofthe piperidine ring is the nitric oxide donor group —ONO₂; and the R₁group linked to the carbon atom at position 5 of the piperidine ring isH, i.e., 2,2,6,6-tetramethyl-3,4-bis(nitrooxy)piperidin-1-yl acetate,2,2,6,6-tetramethyl-3,4-bis(nitrooxy)piperidin-1-yl propionate, and2,2,6,6-tetramethyl-3,4-bis(nitrooxy) piperidin-1-yl isobutyrate (hereinidentified compounds 5b₁, 5b₂ and 5b₃, respectively); (vii) each one ofthe R₁ groups linked to the carbon atoms at positions 3 and 5 of thepiperidine ring is the nitric oxide donor group —CH₂—ONO₂; and the R₁group linked to the carbon atom at position 4 of the piperidine ring isH, i.e., 2,2,6,6-tetramethyl-3,5-bis(nitrooxymethyl)piperidin-1-ylacetate, 2,2,6,6-tetramethyl-3,5-bis(nitrooxymethyl)piperidin-1-ylpropionate, and 2,2,6,6-tetramethyl-3,5-bis(nitrooxymethyl)piperidin-1-yl isobutyrate (herein identifiedcompounds 6a₁, 6a₂ and 6a₃, respectively); (viii) each one of the R₁groups linked to the carbon atoms at positions 3 and 5 of the piperidinering is the nitric oxide donor group —ONO₂; and the R₁ group linked tothe carbon atom at position 4 of the piperidine ring is H, i.e.,2,2,6,6-tetramethyl-3,5-bis(nitrooxy)piperidin-1-yl acetate,2,2,6,6-tetramethyl-3,5-bis(nitrooxy)piperidin-1-yl propionate, and2,2,6,6-tetramethyl-3,5-bis(nitrooxy)piperidin-1-yl isobutyrate (hereinidentified compounds 6b₁, 6b₂ and 6b₃, respectively); (ix) each one ofthe R₁ groups linked to the carbon atoms at positions 3 to 5 of thepiperidine ring is the nitric oxide donor group —CH₂—ONO₂, i.e.,2,2,6,6-tetramethyl-3,4,5-tris(nitrooxymethyl) piperidin-1-yl acetate,2,2,6,6-tetramethyl-3,4,5-tris(nitrooxymethyl) piperidin-1-ylpropionate, and2,2,6,6-tetramethyl-3,4,5-tris(nitrooxymethyl)piperidin-1-yl isobutyrate(herein identified compounds 7a₁, 7a₂ and 7a₃, respectively); or (x)each one of the R₁ groups linked to the carbon atoms at positions 3 to 5of the piperidine ring is the nitric oxide donor group —ONO₂, i.e.,2,2,6,6-tetramethyl-3,4,5-tris(nitrooxy) piperidin-1-yl acetate,2,2,6,6-tetramethyl-3,4,5-tris(nitrooxy)piperidin-1-yl propionate, and2,2,6,6-tetramethyl-3,4,5-tris(nitrooxy)piperidin-1-yl isobutyrate(herein identified compounds 7b₁, 7b₂ and 7b₃, respectively).

In further specific embodiments, the compound used according to themethod of the invention is the compound of formula Ic, i.e., a compoundof the general formula I wherein n is 3, wherein R₂ each is methyl; R₃is methyl, ethyl, or isopropyl; and (i) the R₁ group linked to thecarbon atom at position 3 of the azepane ring is the nitric oxide donorgroup —CH₂—ONO₂; and each one of the R₁ groups linked to the carbonatoms at positions 4 to 6 of the azepane ring is H, i.e.,2,2,7,7-tetramethyl-3-(nitrooxymethyl)azepan-1-yl acetate,2,2,7,7-tetramethyl-3-(nitrooxymethyl)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3-(nitrooxymethyl)azepan-1-yl isobutyrate (hereinidentified compounds 8a₁, 8a₂ and 8a₃, respectively); (ii) the R₁ grouplinked to the carbon atom at position 3 of the azepane ring is thenitric oxide donor group —ONO₂; and each one of the R₁ groups linked tothe carbon atoms at positions 4 to 6 of the azepane ring is H, i.e.,2,2,7,7-tetramethyl-3-(nitrooxy)azepan-1-yl acetate,2,2,7,7-tetramethyl-3-(nitrooxy) azepan-1-yl propionate, and2,2,7,7-tetramethyl-3-(nitrooxy)azepan-1-yl isobutyrate (hereinidentified compounds 8b₁, 8b₂ and 8b₃, respectively); (iii) the R₁ grouplinked to the carbon atom at position 4 of the azepane ring is thenitric oxide donor group —CH₂—ONO₂; and each one of the R₁ groups linkedto the carbon atoms at position 3, 5 and 6 of the azepane ring is H,i.e., 2,2,7,7-tetramethyl-4-(nitrooxymethyl)azepan-1-yl acetate,2,2,7,7-tetramethyl-4-(nitrooxymethyl) azepan-1-yl propionate, and2,2,7,7-tetramethyl-4-(nitrooxymethyl)azepan-1-yl isobutyrate (hereinidentified compounds 9a₁, 9a₂ and 9a₃, respectively); (iv) the R₁ grouplinked to the carbon atom at position 4 of the azepane ring is thenitric oxide donor group —ONO₂; and each one of the R₁ groups linked tothe carbon atoms at position 3, 5 and 6 of the azepane ring is H, i.e.,2,2,7,7-tetramethyl-4-(nitrooxy)azepan-1-yl acetate,2,2,7,7-tetramethyl-4-(nitrooxy)azepan-1-yl propionate, and2,2,7,7-tetramethyl-4-(nitrooxy) azepan-1-yl isobutyrate (hereinidentified compounds 9b₁, 9b₂ and 9b₃, respectively); (v) each one ofthe R₁ groups linked to the carbon atoms at positions 3 and 4 of theazepane ring is the nitric oxide donor group —CH₂—ONO₂; and each one ofthe R₁ groups linked to the carbon atoms at positions 5 and 6 of theazepane ring is H, i.e.,2,2,7,7-tetramethyl-3,4-bis(nitrooxymethyl)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,4-bis(nitrooxymethyl) azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,4-bis(nitrooxymethyl)azepan-1-yl isobutyrate(herein identified compounds 10a₁, 10a₂ and 10a₃, respectively); (vi)each one of the R₁ groups linked to the carbon atoms at positions 3 and4 of the azepane ring is the nitric oxide donor group —ONO₂; and eachone of the R₁ groups linked to the carbon atoms at positions 5 and 6 ofthe azepane ring is H, i.e.,2,2,7,7-tetramethyl-3,4-bis(nitrooxy)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,4-bis(nitrooxy)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,4-bis(nitrooxy) azepan-1-yl isobutyrate (hereinidentified compounds 10b₁, 10b₂ and 10b₃, respectively); (vii) each oneof the R₁ groups linked to the carbon atoms at positions 3 and 5 of theazepane ring is the nitric oxide donor group —CH₂—ONO₂; and each one ofthe R₁ groups linked to the carbon atoms at positions 4 and 6 of theazepane ring is H, i.e.,2,2,7,7-tetramethyl-3,5-bis(nitrooxymethyl)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,5-bis(nitrooxymethyl)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,5-bis(nitrooxymethyl)azepan-1-yl isobutyrate(herein identified compounds 11a₁, 11a₂ and 11a₃, respectively); (viii)each one of the R₁ groups linked to the carbon atoms at positions 3 and5 of the azepane ring is the nitric oxide donor group —ONO₂; and eachone of the R₁ groups linked to the carbon atoms at positions 4 and 6 ofthe azepane ring is H, i.e.,2,2,7,7-tetramethyl-3,5-bis(nitrooxy)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,5-bis(nitrooxy) azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,5-bis(nitrooxy)azepan-1-yl isobutyrate (hereinidentified compounds 11b₁, 11b₂ and 11b₃, respectively); (ix) each oneof the R₁ groups linked to the carbon atoms at positions 3 and 6 of theazepane ring is the nitric oxide donor group —CH₂—ONO₂; and each one ofthe R₁ groups linked to the carbon atoms at positions 4 and 5 of theazepane ring is H, i.e.,2,2,7,7-tetramethyl-3,6-bis(nitrooxymethyl)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,6-bis(nitrooxymethyl)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,6-bis(nitrooxymethyl)azepan-1-yl isobutyrate(herein identified compounds 12a₁, 12a₂ and 12a₃, respectively); (x)each one of the R₁ groups linked to the carbon atoms at positions 3 and6 of the azepane ring is the nitric oxide donor group —ONO₂; and eachone of the R₁ groups linked to the carbon atoms at positions 4 and 5 ofthe azepane ring is H, i.e.,2,2,7,7-tetramethyl-3,6-bis(nitrooxy)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,6-bis(nitrooxy)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,6-bis(nitrooxy)azepan-1-yl isobutyrate (hereinidentified compounds 12b₁, 12b₂ and 12b₃, respectively); (xi) each oneof the R₁ groups linked to the carbon atoms at positions 3 to 5 of theazepane ring is the nitric oxide donor group —CH₂—ONO₂; and the R₁ grouplinked to the carbon atom at position 6 of the azepane ring is H, i.e.,2,2,7,7-tetramethyl-3,4,5-tris(nitrooxymethyl) azepan-1-yl acetate,2,2,7,7-tetramethyl-3,4,5-tris(nitrooxymethyl) azepan-1-yl propionate,and 2,2,7,7-tetramethyl-3,4,5-tris(nitrooxymethyl)azepan-1-ylisobutyrate (herein identified compounds 13a₁, 13a₂ and 13a₃,respectively); (xii) each one of the R₁ groups linked to the carbonatoms at positions 3 to 5 of the azepane ring is the nitric oxide donorgroup —ONO₂; and the R₁ group linked to the carbon atom at position 6 ofthe azepane ring is H, i.e.,2,2,7,7-tetramethyl-3,4,5-tris(nitrooxy)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,4,5-tris(nitrooxy)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,4,5-tris(nitrooxy) azepan-1-yl isobutyrate (hereinidentified compounds 13b₁, 13b₂ and 13b₃, respectively); (xiii) each ofthe R₁ groups linked to the carbon atoms at positions 3, 4 and 6 of theazepane ring is the nitric oxide donor group —CH₂—ONO₂; and the R₁ grouplinked to the carbon atom at position 5 of the azepane ring is H, i.e.,2,2,7,7-tetramethyl-3,4,6-tris(nitrooxymethyl)azepan-1-yl acetate,2,2,7,7-tetramethyl-3,4,6-tris(nitrooxymethyl) azepan-1-yl propionate,and 2,2,7,7-tetramethyl-3,4,6-tris(nitrooxymethyl)azepan-1-ylisobutyrate (herein identified compounds 14a₁, 14a₂ and 14a₃,respectively); (xiv) each of the R₁ groups linked to the carbon atoms atpositions 3, 4 and 6 of the azepane ring is the nitric oxide donor group—ONO₂; and the R₁ group linked to the carbon atom at position 5 of theazepane ring is H, i.e., 2,2,7,7-tetramethyl-3,4,6-tris(nitrooxy)azepan-1-yl acetate, 2,2,7,7-tetramethyl-3,4,6-tris(nitrooxy)azepan-1-ylpropionate, and 2,2,7,7-tetramethyl-3,4,6-tris(nitrooxy)azepan-1-ylisobutyrate (herein identified compounds 14b₁, 14b₂ and 14b₃,respectively); (xv) each of the R₁ groups linked to the carbon atoms atpositions 3 to 6 of the azepane ring is the nitric oxide donor group—CH₂—ONO₂, i.e., 2,2,7,7-tetramethyl-3,4,5,6-tetrakis(nitrooxymethyl)azepan-1-yl acetate, 2,2,7,7-tetramethyl-3,4,5,6-tetrakis(nitrooxymethyl)azepan-1-yl propionate, and2,2,7,7-tetramethyl-3,4,5,6-tetrakis (nitrooxymethyl)azepan-1-ylisobutyrate (herein identified compounds 15a₁, 15a₂ and 15a₃,respectively); or (xvi) each of the R₁ groups linked to the carbon atomsat positions 3 to 6 of the azepane ring is the nitric oxide donor group—ONO₂, i.e., 2,2,7,7-tetramethyl-3,4,5,6-tetrakis(nitrooxy)azepan-1-ylacetate, 2,2,7,7-tetramethyl-3,4,5,6-tetrakis(nitrooxy) azepan-1-ylpropionate, and 2,2,7,7-tetramethyl-3,4,5,6-tetrakis(nitrooxy)azepan-1-yl isobutyrate (herein identified compounds 15b₁,15b₂ and 15b₃, respectively).

In still other specific embodiments, the compound of the invention isthe compound of formula Ia, wherein R₂ each is methyl; R₃ is methyl,ethyl, or isopropyl; and (i) the R₁ group linked to the carbon atom atposition 3 of the pyrrolidine ring is the nitric oxide donor group—CH₂—ONO₂; and the R₁ group linked to the carbon atom at position 4 ofthe pyrrolidine ring is —CONH₂, i.e.,2,2,5,5-tetramethyl-3-(nitrooxymethyl)-4-carbamoyl-pyrrolidin-1-ylacetate,2,2,5,5-tetramethyl-3-(nitrooxymethyl)-4-carbamoyl-pyrrolidin-1-ylpropionate, and2,2,5,5-tetramethyl-3-(nitrooxymethyl)-4-carbamoyl-pyrrolidin-1-ylisobutyrate (herein identified compounds 16a₁, 16a₂ and 16a₃,respectively); or (ii) the R₁ group linked to the carbon atom atposition 3 of the pyrrolidine ring is the nitric oxide donor group—ONO₂; and the R₁ group linked to the carbon atom at position 4 of thepyrrolidine ring is —CONH₂, i.e.,2,2,5,5-tetramethyl-3-(nitrooxy)-4-carbamoyl-pyrrolidin-1-yl acetate,2,2,5,5-tetramethyl-3-(nitrooxy)-4-carbamoyl-pyrrolidin-1-yl propionate,and 2,2,5,5-tetramethyl-3-(nitrooxy)-4-carbamoyl-pyrrolidin-1-ylisobutyrate (herein identified compounds 16b₁, 16b₂ and 16b₃,respectively).

In yet other specific embodiments, the compound of the invention is thecompound of formula Ib, wherein R₂ each is methyl; R₃ is methyl, ethyl,or isopropyl; and (i) the R₁ group linked to the carbon atom at position3 of the piperidine ring is the nitric oxide donor group —CH₂—ONO₂; theR₁ group linked to the carbon atom at position 4 of the piperidine ringis —COOH; and the R₁ group linked to the carbon atoms at position 5 ofthe piperidine ring is H, i.e.,2,2,6,6-tetramethyl-3-(nitrooxymethyl)-4-carboxy-piperidin-1-yl acetate,2,2,6,6-tetramethyl-3-(nitrooxymethyl)-4-carboxy-piperidin-1-ylpropionate, and2,2,6,6-tetramethyl-3-(nitrooxymethyl)-4-carboxy-piperidin-1-ylisobutyrate (herein identified compounds 17a₁, 17a₂ and 17a₃,respectively); or (ii) the R₁ group linked to the carbon atom atposition 3 of the piperidine ring is the nitric oxide donor group —ONO₂;the R₁ group linked to the carbon atom at position 4 of the piperidinering is —COOH; and the R₁ group linked to the carbon atoms at position 5of the piperidine ring is H, i.e.,2,2,6,6-tetramethyl-3-(nitrooxy)-4-carboxy-piperidin-1-yl acetate,2,2,6,6-tetramethyl-3-(nitrooxy)-4-carboxy-piperidin-1-yl propionate,and 2,2,6,6-tetramethyl-3-(nitrooxy)-4-carboxy-piperidin-1-ylisobutyrate (herein identified compounds 17b₁, 17b₂ and 17b₃,respectively).

TABLE 2 Compounds of the general formula I, herein identified 1a₁₋₃ to15a₁₋₃*

1a₁₋₃

2a₁₋₃

3a₁₋₃

4a₁₋₃

5a₁₋₃

6a₁₋₃

7a₁₋₃

8a₁₋₃

9a₁₋₃

10a₁₋₃

11a₁₋₃

12a₁₋₃

13a₁₋₃

14a₁₋₃

15a₁₋₃ *Similar compounds in which each one of the nitric oxide donorgroups is —ONO₂ rather than —CH₂—ONO₂ are herein identified compounds1b₁₋₃ to 15b₁₋₃

In still a further specific embodiment, the compound of the invention isthe compound of formula Ib, wherein R₂ each is methyl; R₃ is methyl,ethyl, or isopropyl; the R₁ group linked to the carbon atom at position4 of the piperidine ring is the nitric oxide donor group—O—CH₂—CH(ONO₂)CH₂—ONO₂; and each of the R₁ groups linked to the carbonatoms at positions 3 and 5 of the piperidine ring is H, i.e.,2,2,6,6-tetramethyl-4-(2,3-bis(nitrooxy)propyloxy)-piperidin-1-ylacetate,2,2,6,6-tetramethyl-4-(2,3-bis(nitrooxy)propyloxy)-piperidin-1-ylpropionate, and2,2,6,6-tetramethyl-4-(2,3-bis(nitrooxy)propyloxy)-piperidin-1-ylisobutyrate (herein identified compounds 18₁, 18₂ and 18₃,respectively).

The compounds of the present invention may be synthesized according toany technology or procedure known in the art, e.g., as described withrespect to compounds 1a₁, 1a₂ and 1a₃ in the Examples sectionhereinafter.

TABLE 3 Compounds of the formula I, herein identified 16a₁₋₃*, 17a₁₋₃*and 18₁₋₃

16a₁₋₃

17a₁₋₃

18₁₋₃ *Similar compounds in which each one of the nitric oxide donorgroup is —ONO₂ rather than —CH₂—ONO₂ are herein identified compounds16b₁₋₃ and 17b₁₋₃

The compounds of the general formula I may have one or more asymmetriccenters, and may accordingly exist both as enantiomers, i.e., opticalisomers (R, S, or racemate, wherein a certain enantiomer may have anoptical purity of 90%, 95%, 99% or more) and as diastereoisomers.Specifically, those chiral centers may be, e.g., in each one of thecarbon atoms of the 1-pyrrolidinyl ester derivative, 1-piperidinyl esterderivative; and 1-azepanyl ester derivative of the general formulas Ia,Ib and Ic, respectively. It should be understood that the presentinvention encompasses all such enantiomers, isomers and mixturesthereof, as well as pharmaceutically acceptable salts and solvatesthereof.

Optically active forms of the compounds of the general formula I may beprepared using any method known in the art, e.g., by resolution of theracemic form by recrystallization techniques; by chiral synthesis; byextraction with chiral solvents; or by chromatographic separation usinga chiral stationary phase. A non-limiting example of a method forobtaining optically active materials is transport across chiralmembranes, i.e., a technique whereby a racemate is placed in contactwith a thin membrane barrier, the concentration or pressure differentialcauses preferential transport across the membrane barrier, andseparation occurs as a result of the non-racemic chiral nature of themembrane that allows only one enantiomer of the racemate to passthrough. Chiral chromatography, including simulated moving bedchromatography, can also be used. A wide variety of chiral stationaryphases are commercially available.

The compounds of the present invention are prodrugs of the corresponding1-pyrrolidinyloxy, 1-piperidinyloxy, and 1-azepanyloxy derivativesdisclosed in U.S. Pat. Nos. 6,448,267, 6,455,542 and 6,759,430, and arethus expected to be effective in all those clinical indications whereinthose compounds are of benefit, i.e., in the prevention, treatment ormanagement of any condition associated with oxidative stress orendothelial dysfunction. Such conditions include, without being limitedto, retinal ischemia reperfusion injury; acute anterior ischemic opticneuropathy; central retinal artery occlusion; hemolytic diseases,including spherocytosis, G6PD deficiency, sickle cell disease,thalassemias, and paroxysmal nocturnal hemoglobinuria; diabetesmellitus, including diabetic wounds and diabetic retinopathy,nephropathy, and cardiovascular disease; cardiovascular diseases such asischemic heart disease, angina pectoris, myocardial ischemia reperfusioninjury and infarction, acute and chronic limb ischemia reperfusioninjury, congestive heart failure (CHF), atherosclerosis, peripheralarterial hypertension cardiac arrhythmias, idiopathic pulmonaryhypertension, pulmonary hypertension associated with idiopathicpulmonary fibrosis, pulmonary hypertension associated with hemolyticdisease, primary pulmonary hypertension of the newborn, pulmonaryhypertension secondary to congenital diaphragmatic hernia and meconiumaspiration; pulmonary hypertension secondary to congenital heartdisease; pulmonary hypertension secondary to mitral regurgitation,atrial or ventricular septal defect; contrast-induced nephropathy;asthma; trauma; hypovolemic, neurogenic, or septic shock; idiopathicerectile dysfunction; erectile dysfunction secondary to radical nervesparing prostatectomy; toxicant-induced inhalational lung injury;chlorine inhalational lung injury; neurotoxicity; neurodegenerative andneurological disorders including Alzheimer and Parkinson's diseases,amyotrophic lateral sclerosis, multiple sclerosis, convulsive (seizure)disorders, AIDS-dementia and disorders which involve processes oflearning and memory; glaucoma and intraocular hypertension; disorders ofgastric secretions, relaxation and peristalsis of the intestinal tract(including sphincters); drug and disease-induced nephropathies;pathological (premature) and physiological uterine contractions;cellular defense impairment; endothelial dysfunction-induced diseases;insulin-resistance in diabetes; pregnancy-induced hypertension;cerebrovascular diseases; aggregation disorders; and female sexualdysfunction, including vaginal dryness.

The compounds of the present invention allow for more concentratedliquid delivery of the compounds to which they are converted underphysiological conditions, as particularly shown with respect to R-107that is, upon exposure to plasma, converted to R-100 (Example 4hereinafter). The possibility of delivering smaller volume of the lattermay be advantageous in those clinical scenarios where the volume ofadministration is limiting, e.g., CHF, intramuscular and subcutaneousinjections, and all topical applications. As further found whilereducing the present invention into practice, the compounds of thepresent invention are converted to, and release, their corresponding1-pyrrolidinyloxy, 1-piperidinyloxy and 1-azepanyloxy derivatives, overa defined period of time in biological solutions and tissues. As such,these compounds thereby provide a sustained release of theircorresponding 1-pyrrolidinyloxy, 1-piperidinyloxy and 1-azepanyloxyderivatives that is reflected in a lower Cmax in the blood. This may beadvantageous in those settings where rapid administration of the latteris undesirable and a sustained release is preferred.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of the general formula I as definedabove, or an enantiomer, diastereomer, racemate, or pharmaceuticallyacceptable salt or solvate thereof, herein also identified as the activeagent, and a pharmaceutically acceptable carrier. Particular suchpharmaceutical compositions comprise, as an active agent, a compoundselected from the compounds of Tables 2-3 above, e.g., compound 1a₁, 1a₂or 1a₃, or an enantiomer, diastereomer, racemate, or pharmaceuticallyacceptable salt or solvate thereof.

The pharmaceutical compositions of the present invention are useful forprevention, treatment or management of a disease, disorder or conditionassociated with oxidative stress or endothelial dysfunction.

WO 2012/093383 discloses methods and compositions for treatment ofsepsis and conditions associated therewith using the 1-pyrrolidinyloxy,1-piperidinyloxy and 1-azepanyloxy derivatives disclosed in U.S. Pat.Nos. 6,448,267, 6,455,542 and 6,759,430, while particularly exemplifyingR-100. Examples 5 and 6 hereinafter describe protocols showing theefficacy of R-107 as a therapeutic agent in mice exposed to a lethaldose of E. coli lipopolysaccharide (LPS), and on pulmonary function insheep suffering from Pseudomonas aeruginosa-induced septic pneumonia.

In certain embodiments, the pharmaceutical composition of the inventionis thus used for treatment of sepsis, particularly caused bymicroorganisms or products thereof, and conditions associated therewith.The sepsis may be caused by Gram negative bacteria, e.g., Escherichiacoli, Pseudomonas aeruginosa, Serratia species, Salmonella species,Shigella species, Enterobacter species, Citrobacter species, Proteusspecies, and Klebsiella species; Gram-positive cocci, e.g., Pneumococcalspecies, Enterococcal species, Staphylococcal species, and Streptococcalspecies; certain fungi and yeast, Rickettsial species, Plasmodialspecies, Clostridial species, or viruses; or Gram-positive bacterialtoxins, including toxic shock syndrome toxins. In particular suchembodiments, treatment with the compounds or pharmaceutical compositionsof the invention is aimed at inhibiting development of sepsis-relatedcoagulopathy.

The term “treatment” as used herein with respect to sepsis andconditions associated therewith refers to administration of an activeagent after the onset of symptoms of sepsis, regardless of the cause forthat medical condition. According to the invention, administration ofsaid active agent for treatment of sepsis and conditions associatedtherewith is aimed at inhibiting, i.e., limiting or reducing, medicalconditions resulting from the systemic infection, most particularlypulmonary arterial hypertension, pulmonary shunt, and loss of pulmonarycompliance, and in certain cases also development of sepsis-relatedcoagulopathy.

WO 2011/092690 discloses methods and compositions for prevention,treatment, or management of PAH using the 1-pyrrolidinyloxy,1-piperidinyloxy and 1-azepanyloxy derivatives disclosed in U.S. Pat.Nos. 6,448,267, 6,455,542 and 6,759,430, while particularly exemplifyingR-100. Examples 7-9 hereinafter describe protocols showing the efficacyof R-107 as a therapeutic agent in a rat model of PAH, and onmonocrotaline (MCT)-induced pulmonary vascular remodeling; a lamb modelof pulmonary hypertension of the newborn; and a minipig model ofperipheral hypertension, respectively.

In certain embodiments, the pharmaceutical composition of the inventionis thus used for prevention, treatment or management of PH, particularlyPAH, PH associated with a left heart disease, PH associated with a lungdisease and/or hypoxemia, or PH due to a chronic thrombotic and/orembolic disease. The pharmaceutical composition of the invention can beused for treatment any form of PH including, but not limited to, mild,i.e., associated with an increase of up to 30, more particularly 20-30,mmHg in mean pulmonary arterial pressure (MPAP) at rest; moderate, i.e.,associated with an increase of 30-39 mmHg in MPAP at rest; and severe,i.e., associated with an increase of 40 mmHg or more in MPAP at rest.

The PAH may be idiopathic PAH; familial PAH; PAH associated withcollagen vascular disease; PAH associated with congenital heartdisorders; PAH associated with HIV infection; PAH associated with venousor capillary diseases; PAH associated with thyroid disorders, glycogenstorage disease, Gaucher's disease, hemoglobinopathies, ormyeloproliferative disorders; PAH associated with either smokeinhalation or combined smoke inhalation and burn injury; PAH associatedwith aspiration; PAH associated with ventilator injury; PAH associatedwith pneumonia; PAH associated with Adult Respiratory Distress Syndrome;persistent PH of the newborn; neonatal respiratory distress syndrome ofprematurity; neonatal meconium aspiration; neonatal diaphragmatichernia; pulmonary capillary hemangiomatosis; or pulmonary veno-occlusivedisease. The left heart disease may be a left sided atrial orventricular disease; or a valvular disease. The lung disease may bechronic obstructive pulmonary disease; an interstitial lung disease;sleep-disordered breathing; an alveolar hypoventilation disorder;chronic exposure to high altitude; or a developmental lung abnormality.The chronic thrombotic and/or embolic disease may be thromboembolicobstruction of distal or proximal pulmonary arteries; or anon-thrombotic pulmonary embolism of, e.g., tumor cells or parasites.

Many of the diseases, disorders and conditions listed above can beassociated with increased risk for PH, wherein particular examplesinclude congenital heart disease, e.g., Eisenmenger syndrome; left heartdisease; pulmonary venous disease, e.g., fibrosis tissue narrowing oroccluding pulmonary veins and venules; pulmonary arterial disease;diseases causing alveolar hypoxia; fibrotic lung diseases; Williamssyndrome; subjects with intravenous drug abuse injury; pulmonaryvasculitis such as Wegener's, Goodpasture's, and Churg-Strausssyndromes; emphysema; chronic bronchitis; kyphoscoliosis; cysticfibrosis; obesity-hyper-ventilation and sleep apnea disorders; pulmonaryfibrosis; sarcoidosis; silocosis; CREST (calcinosis cutis, Raynaudphenomenon; esophageal motility disorder; sclerodactyly, andteleangiectasia) and other connective tissue diseases. For example, asubject who possesses a bone morphogenetic protein receptor E (BMPR2)mutation has a 10-20% lifetime risk of acquiring FPAH, and subjects withhereditary hemorrhagic telangiectasa, particularly those carryingmutations in ALKI, were also identified as being at risk for IPAH. Riskfactors and diagnostic criteria for PH are described in McGoon et al.,Chest, 2004, 126, 14S-34S.

The terms “treatment” and “prevention” as used herein with respect to PHrefer to administration of an active agent after the onset of symptomsof PH in any of its forms, or prior to the onset of symptoms,particularly to patients at risk for PH, respectively. The term“management” as used herein with respect to PH refers to prevention ofrecurrence of PH in a patient previously suffered from PH.

WO 2013/005216 discloses methods and compositions for prevention andtreatment of renal ischemia-reperfusion injury using the1-pyrrolidinyloxy, 1-piperidinyloxy and 1-azepanyloxy derivativesdisclosed in U.S. Pat. Nos. 6,448,267, 6,455,542 and 6,759,430, whileparticularly exemplifying R-100. Example 10 hereinafter describes aprotocol showing the efficacy of R-107 as a therapeutic agent in a ratmodel of renal ischemia-reperfusion injury. In certain embodiments, thepharmaceutical composition of the invention is thus used for preventionor treatment of renal ischemia-reperfusion injury.

The term “renal ischemia” refers to a deficiency of blood flow in one orboth kidneys, or nephrons, usually due to functional constriction oractual obstruction of a blood vessel or surgical removal of the kidney.Renal ischemia may result from various medical conditions including,without being limited to, hemorrhagic shock, septic shock, asphyxia alsoknown as asphyxiation, cardiac arrest also known as cardiopulmonaryarrest or circulatory arrest, respiratory arrest, respiratory failure,cardiogenic shock, aortic aneurysm, aortic aneurysm surgery,hypotension, dehydration, spinal shock, trauma, cadaveric renaltransplantation, living related donor renal transplantation, livertransplantation, a liver disease, drug-induced renal ischemia,hydronephrosis, urethral obstruction, cardiopulmonary bypass surgery,radiocontrast administration, endovascular renal artery catheterization,renovascular stenosis, renal artery thrombosis, ureteral obstruction,hypoxia, and hypoxemia. The term “renal ischemia-reperfusion injury”refers to the damage caused to the kidney(s) when blood supply returnsto the tissue after a period of renal ischemia. Renalischemia-reperfusion injury is characterized by renal dysfunction andtubular damages, and considered as a major cause of acute renal failurethat may also be involved in the development and progression of someforms of chronic kidney disease. In general, the absence of oxygen andnutrients from blood during the ischemic period creates a condition inwhich the restoration of circulation results in inflammation andoxidative damage through the induction of oxidative stress rather thanrestoration of normal function.

The term “treatment” as used herein with respect to renalischemia-reperfusion injury refers to administration of an active agentafter the onset of symptoms of renal ischemia-reperfusion injury, i.e.,after blood supply to the ischemic tissue has been renewed, regardlessof the cause for the renal ischemia. The term “prevention” as usedherein with respect to renal ischemia-reperfusion injury refers toadministration of said active agent prior to the onset of symptoms,i.e., either prior to the onset of renal ischemia or following the onsetof renal ischemia but prior to reperfusion, and it is mainly relevant incases wherein the renal ischemia and/or reperfusion is/are associatedwith a surgical intervention, e.g., with aortic aneurysm surgery,cadaveric renal transplantation, living related donor renaltransplantation, liver transplantation, cardiopulmonary bypass surgery,or endovascular renal artery catheterization. According to theinvention, administration of said active agent either for treatment orprevention of renal ischemia-reperfusion injury is aimed at inhibiting,i.e., limiting or reducing, renal dysfunction, PMN infiltration into therenal parenchyma, and histological damage, i.e., tubular necrosis.

Examples 11-12 hereinafter describe protocols showing the efficacy ofR-107 as a therapeutic agent in murine and porcine models of retinalischemia-reperfusion injury. In certain embodiments, the pharmaceuticalcomposition of the invention is thus used for treatment of retinalischemia-reperfusion injury.

The term “retinal ischemia” refers to those conditions wherein the bloodsupply to the retinal cells is impaired, resulting in a deficiency ofoxygenation to retinal tissue. The term “retinal ischemia-reperfusioninjury” refers to those conditions in which retinal ischemia is followedby improved blood flow (so-called “reperfusion”), which thus increasesoxidant formation and consequently induces tissue injury.

The term “treatment” as used herein with respect to retinalischemia-reperfusion injury refers to administration of an active agentafter the onset of symptoms of retinal ischemia-reperfusion injury,i.e., after blood supply to the ischemic tissue has been renewed,regardless of the cause for the retinal ischemia. According to theinvention, administration of said active agent for treatment of retinalischemia-reperfusion injury is aimed at inhibiting, i.e., limiting orreducing, the extent of retinal damage and ultimately loss of vision.

Example 13 hereinafter describes a protocol showing the efficacy ofR-107 as a therapeutic agent in a rat model of myocardialischemia-reperfusion injury. In certain embodiments, the pharmaceuticalcomposition of the invention is thus used for prevention or treatment ofmyocardial ischemia-reperfusion injury.

The term “myocardial ischemia” refers to those conditions in which thereis a mismatch of oxygen supply and demand in the myocardium. The term“myocardial ischemia-reperfusion injury” refers to those settings whererevascularization of an ischemic region of the heart is accompanied byan increased flux of free radicals and the subsequent evolution ofmyocardial tissue damage.

The term “treatment” as used herein with respect to myocardialischemia-reperfusion injury refers to administration of an active agentafter the onset of symptoms of myocardial ischemia-reperfusion injury,i.e., after blood supply to the ischemic tissue has been renewed,regardless of the cause for the myocardial ischemia. The term“prevention” as used herein with respect to myocardialischemia-reperfusion injury refers to administration of said activeagent prior to the onset of symptoms, i.e., either prior to the onset ofmyocardial ischemia or following the onset of myocardial ischemia butprior to reperfusion, and it is mainly relevant in cases wherein anacute therapeutic revascularization event is undertaken, such asangioplasty or thrombolysis. According to the invention, administrationof said active agent either for treatment or prevention of myocardialischemia-reperfusion injury is aimed at inhibiting, i.e., limiting orreducing, the extent of devitalized myocardium and the appearance ofarrhythmias.

Example 14 hereinafter describes a protocol showing the efficacy ofR-107 as a therapeutic agent in a minipig model of acute limbischemia-reperfusion injury. In certain embodiments, the pharmaceuticalcomposition of the invention is thus used for prevention or treatment oflimb ischemia-reperfusion injury.

The term “limb ischemia” refers to a mismatch in the supply and demandof oxygen to an extremity. The term “limb ischemia-reperfusion injury”refers to the injury resulting from an acute restoration of blood flowto a previously ischemic extremity.

The term “treatment” as used herein with respect to limbischemia-reperfusion injury refers to administration of an active agentafter the onset of symptoms of limb ischemia-reperfusion injury, i.e.,after blood supply to the ischemic tissue has been renewed, regardlessof the cause for the limb ischemia. The term “prevention” as used hereinwith respect to limb ischemia-reperfusion injury refers toadministration of said active agent prior to the onset of symptoms,i.e., either prior to the onset of limb ischemia or following the onsetof limb ischemia but prior to reperfusion, and it is mainly relevant incases wherein restoration of blood flow to the affected limb takes placewithin 6-12 hours of acute ischemia. According to the invention,administration of said active agent either for treatment or preventionof limb ischemia-reperfusion injury is aimed at inhibiting, i.e.,limiting or reducing, muscle damage, muscle weakness, limb necrosis andgangrene, limb amputation, myoglobinuria, renal failure, compartmentsyndrome, hyperkalemia, acute respiratory distress syndrome, andcirculatory shock.

Example 15 hereinafter describes a protocol showing the efficacy ofR-107 as a therapeutic agent in a rat model of contrast-inducednephropathy. In certain embodiments, the pharmaceutical composition ofthe invention is thus used for prevention or treatment ofcontrast-induced nephropathy.

The term “contrast-induced nephropathy” refers to the impairment ofrenal morphology and function resulting from the acute administration ofa radio-opaque dye for purposes of angiography, with said nephropathytypically occurring in the setting of underlying renal disease, asreflected by a reduced glomerular filtration rate and tubular functionand elevated serum blood urea nitrogen (BUN) and/or creatinine.

The terms “treatment” and “prevention” as used herein with respect tocontrast-induced nephropathy refer to administration of an active agentafter the onset of symptoms of contrast-induced nephropathy, or prior tothe onset of symptoms, respectively. According to the invention,administration of said active agent either for treatment or preventionof contrast-induced nephropathy is aimed at ensuring the vitality of thekidney, as reflected by morphologic assessment and functional measuresof glomerular filtration and tubular behavior, such as serum BUN andcreatinine.

WO 2013/190497 discloses methods and compositions for treatment of CILIusing, inter alia, the 1-pyrrolidinyloxy, 1-piperidinyloxy and1-azepanyloxy derivatives disclosed in U.S. Pat. Nos. 6,448,267,6,455,542 and 6,759,430, while particularly exemplifying R-100. Example16 hereinafter shows the efficacy of R-107 as a therapeutic agent in amurine Cl₂ exposure model.

In certain embodiments, the pharmaceutical composition of the inventionis thus used for treatment of an inflammatory disease of the lung causedby inhalation of a toxic agent or an irritant. In one particular suchembodiment, the toxic agent is Cl₂, and the pharmaceutical compositionis used for treatment of CILI. In another particular such embodiments,the toxic agent is the chemical warfare agent phosgene or diphosgene, orthe irritant is smoke.

The term “treatment”, as used herein with respect to an inflammatorydiseases of the lung caused by inhalation of a toxic agent or anirritant, refers to administration of an active agent after exposure tosaid toxic agent or irritant and following the onset of symptoms of saidinflammatory disease, so as to ameliorate the effects of said toxicagent or irritant on the lungs. According to the invention,administration of said active agent for treatment of CILI is aimed atreducing pulmonary edema and pulmonary shunt, diminishing PMNinfiltration into the lung parenchyma, inhibiting a loss in pulmonarycompliance, improving oxygenation, and decreasing carbon dioxideretention.

Example 17 hereinafter describes a protocol showing the efficacy ofR-107 as a therapeutic agent in a rat model of erectile dysfunction. Incertain embodiments, the pharmaceutical composition of the invention isthus used for treatment of erectile dysfunction.

The term “erectile dysfunction” refers to the inability of a male toproduce an erection that allows for reliable and effective penetrationof the vagina. The term “treatment” as used herein with respect toerectile dysfunction refers to administration of an active agent afterthe onset of symptoms of erectile dysfunction. According to theinvention, administration of said active agent for treatment of erectiledysfunction is aimed at improving erectile quality and therebyfacilitating successful vaginal penetration.

The pharmaceutical compositions of the present invention can be providedin a variety of formulations, e.g., in a pharmaceutically acceptableform and/or in a salt form, as well as in a variety of dosages.

In one embodiment, the pharmaceutical composition of the presentinvention comprises a non-toxic pharmaceutically acceptable salt of acompound of the general formula I. Suitable pharmaceutically acceptablesalts include acid addition salts such as, without being limited to, themesylate salt, the maleate salt, the fumarate salt, the tartrate salt,the hydrochloride salt, the hydrobromide salt, the esylate salt, thep-toluenesulfonate salt, the benzenesulfonate salt, the benzoate salt,the acetate salt, the phosphate salt, the sulfate salt, the citratesalt, the carbonate salt, and the succinate salt. Additionalpharmaceutically acceptable salts include salts of ammonium (NH₄ ⁺) oran organic cation derived from an amine of the formula R₄N⁺, whereineach one of the Rs independently is selected from H, C₁-C₂₂, preferablyC₁-C₆ alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl,and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl,pyrimidinyl, and the like, or two of the Rs together with the nitrogenatom to which they are attached form a 3-7 membered ring optionallycontaining a further heteroatom selected from N, S and O, such aspyrrolydine, piperidine and morpholine. Furthermore, where the compoundsof the general formula I carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may include metal salts suchas alkali metal salts, e.g., lithium, sodium or potassium salts, andalkaline earth metal salts, e.g., calcium or magnesium salts.

Further pharmaceutically acceptable salts include salts of a cationiclipid or a mixture of cationic lipids. Cationic lipids are often mixedwith neutral lipids prior to use as delivery agents. Neutral lipidsinclude, but are not limited to, lecithins; phosphatidylethanolamine;diacyl phosphatidylethanolamines such as dioleoylphosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine,palmitoyloleoyl phosphatidylethanolamine and distearoylphosphatidylethanolamine; phosphatidylcholine; diacylphosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoylphosphatidylcholine; phosphatidylglycerol; diacyl phosphatidylglycerolssuch as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglyceroland distearoyl phosphatidylglycerol; phosphatidylserine; diacylphosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine;and diphosphatidylglycerols; fatty acid esters; glycerol esters;sphingolipids; cardiolipin; cerebrosides; ceramides; and mixturesthereof. Neutral lipids also include cholesterol and other 3βhydroxy-sterols.

Examples of cationic lipid compounds include, without being limited to,Lipofectin® (Life Technologies, Burlington, Ontario) (1:1 (w/w)formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride anddioleoylphosphatidyl-ethanolamine); Lipofectamine™ (Life Technologies,Burlington, Ontario) (3:1 (w/w) formulation of polycationic lipid2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-iumtrifluoroacetateand dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (LifeTechnologies, Burlington, Ontario) (Lipofectamine and Plus reagent),Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationiclipid), Effectene (Qiagen, Mississauga, Ontario) (Non liposomal lipidformulation), Metafectene (Biontex, Munich, Germany) (Polycationiclipid), Eu-fectins (Promega Biosciences, San Luis Obispo, Calif.)(ethanolic cationic lipids numbers 1 through 12: C₅₂H₁₀₆N₆O₄.4CF₃CO₂H,C₈₈H₁₇₈N₈O₄S₂.4CF₃CO₂H, C₄₀H₈₄NO₃P.CF₃CO₂H, C₅₀H₁₀₃N₇O₃.4CF₃CO₂H,C₅₅H₁₁₆N₈O₂.6CF₃CO₂H, C₄₉H₁₀₂N₆O₃.4CF₃CO₂H, C₄₄H₈₉N₅O₃.2CF₃CO₂H,C₁₀₀H₂₀₆N₁₂O₄S₂.8CF₃CO₂H, C₁₆₂H₃₃₀N₂₂O₉.13CF₃CO₂H, C₄₃H₈₈N₄O₂.2CF₃CO₂H,C₄₃H₈₈N₄O₃.2CF₃CO₂H, C₄₁H₇₈NO₈P); Cytofectene (Bio-Rad, Hercules,Calif.) (mixture of a cationic lipid and a neutral lipid), GenePORTER®(Gene Therapy Systems, San Diego, Calif.) (formulation of a neutrallipid (Dope) and a cationic lipid) and FuGENE 6 (Roche MolecularBiochemicals, Indianapolis, Ind.) (Multi-component lipid basednon-liposomal reagent).

The pharmaceutically acceptable salts of the present invention may beformed by conventional means, e.g., by reacting a free base form of theactive agent, i.e., the compound of the general formula I, with one ormore equivalents of the appropriate acid in a solvent or medium in whichthe salt is insoluble, or in a solvent such as water which is removed invacuo or by freeze drying, or by exchanging the anion/cation of anexisting salt for another anion/cation on a suitable ion exchange resin.

The present invention encompasses solvates of the compounds of thegeneral formula I as well as salts thereof, e.g., hydrates.

The pharmaceutical compositions provided by the present invention may beprepared by conventional techniques, e.g., as described in Remington:The Science and Practice of Pharmacy, 19^(th) Ed., 1995. Thecompositions can be prepared, e.g., by uniformly and intimately bringingthe active agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into the desired formulation. The compositions may be in liquid,solid or semisolid form and may further include pharmaceuticallyacceptable fillers, carriers, diluents or adjuvants, and other inertingredients and excipients. In one embodiment, the pharmaceuticalcomposition of the present invention is formulated as nanoparticles.

The compositions can be formulated for any suitable route ofadministration, but they are preferably formulated for parenteral, e.g.,intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal,intrapleural, intratracheal, subcutaneous, or topical administration, aswell as for inhalation. Pharmaceutical compositions formulated forintramuscular injections may be suitable, inter alia, for emergent usesuch as in CILI; and pharmaceutical compositions formulated for topicaladministration may be suitable, inter alia, for treatment of skin ulcersand wounds, elevated intraocular pressure and uveitis (by application tothe cornea), and erectile dysfunction (by application to the penile), aswell as for increasing genital lubrication (by vaginal application). Thedosage will depend on the state of the patient, and will be determinedas deemed appropriate by the practitioner.

The pharmaceutical composition of the invention may be in the form of asterile injectable aqueous or oleagenous suspension, which may beformulated according to the known art using suitable dispersing, wettingor suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Acceptable vehicles and solvents that maybe employed include, without limiting, water, Ringer's solution,polyethylene glycol (PEG), 2-hydroxypropyl-β-cyclodextrin (HPCD),Tween-80, and isotonic sodium chloride solution.

Pharmaceutical compositions according to the present invention, whenformulated for inhalation, may be administered utilizing any suitabledevice known in the art, such as metered dose inhalers, liquidnebulizers, dry powder inhalers, sprayers, thermal vaporizers,electrohydrodynamic aerosolizers, and the like.

Pharmaceutical compositions according to the present invention, whenformulated for administration route other than parenteraladministration, may be in a form suitable for oral use, e.g., astablets, troches, lozenges, aqueous, or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs.

Pharmaceutical compositions intended for oral administration should beformulated so as to inhibit the release of the active agent in thestomach, i.e., delay the release of the active agent until at least aportion of the dosage form has traversed the stomach, in order to avoidthe acidity of the gastric contents from hydrolyzing the active agent toits highly water insoluble form, i.e., its corresponding1-pyrrolidinyloxy, 1-piperidinyloxy or 1-azepanyloxy derivative.Particular such compositions are those wherein the active agent iscoated by a pH-dependent enteric-coating polymer. Examples ofpH-dependent enteric-coating polymer include, without being limited to,Eudragit® S (poly(methacrylicacid, methylmethacrylate), 1:2), Eudragit®L 55 (poly (methacrylicacid, ethylacrylate), 1:1), Kollicoat®(poly(methacrylicacid, ethylacrylate), 1:1), hydroxypropylmethylcellulose phthalate (HPMCP), alginates, carboxymethylcellulose,and combinations thereof. The pH-dependent enteric-coating polymer maybe present in the composition in an amount from about 10% to about 95%by weight of the entire composition.

Pharmaceutical compositions intended for oral administration may beprepared according to any method known to the art for the manufacture ofpharmaceutical compositions and may further comprise one or more agentsselected from sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients, whichare suitable for the manufacture of tablets. These excipients may be,e.g., inert diluents such as calcium carbonate, sodium carbonate,lactose, calcium phosphate, or sodium phosphate; granulating anddisintegrating agents, e.g., corn starch or alginic acid; bindingagents, e.g., starch, gelatin or acacia; and lubricating agents, e.g.,magnesium stearate, stearic acid, or talc. The tablets may be eitheruncoated or coated utilizing known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed. They may also be coated using the techniques described in theU.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotictherapeutic tablets for control release. The pharmaceutical compositionof the invention may also be in the form of oil-in-water emulsion.

The pharmaceutical compositions of the invention may be formulated forcontrolled release of the active agent. Such compositions may beformulated as controlled-release matrix, e.g., as controlled-releasematrix tablets in which the release of a soluble active agent iscontrolled by having the active diffuse through a gel formed after theswelling of a hydrophilic polymer brought into contact with dissolvingliquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymershave been described as capable of forming such gel, e.g., derivatives ofcellulose, in particular the cellulose ethers such as hydroxypropylcellulose, hydroxymethyl cellulose, methylcellulose or methylhydroxypropyl cellulose, and among the different commercial grades ofthese ethers are those showing fairly high viscosity. In otherconfigurations, the compositions comprise the active agent formulatedfor controlled release in microencapsulated dosage form, in which smalldroplets of the active agent are surrounded by a coating or a membraneto form particles in the range of a few micrometers to a fewmillimeters.

Another contemplated formulation is depot systems, based onbiodegradable polymers, wherein as the polymer degrades, the activeingredient is slowly released. The most common class of biodegradablepolymers is the hydrolytically labile polyesters prepared from lacticacid, glycolic acid, or combinations of these two molecules. Polymersprepared from these individual monomers include poly (D,L-lactide)(PLA), poly (glycolide) (PGA), and the copolymer poly(D,L-lactide-co-glycolide) (PLG).

In a further aspect, the present invention relates to a compound of thegeneral formula I as defined above, or an enantiomer, diastereomer,racemate, or pharmaceutically acceptable salt or solvate thereof, foruse in prevention, treatment or management of a disease, disorder orcondition associated with oxidative stress or endothelial dysfunction.

In yet another aspect, the present invention relates to use of acompound of the general formula I as defined above, or an enantiomer,diastereomer, racemate, or pharmaceutically acceptable salt or solvatethereof, for the preparation of a pharmaceutical composition forprevention, treatment or management of a disease, disorder or conditionassociated with oxidative stress or endothelial dysfunction.

In still another aspect, the present invention relates to a method forprevention, treatment or management of a disease, disorder or conditionassociated with oxidative stress or endothelial dysfunction in anindividual in need thereof, comprising administering to said individualan effective amount of a compound of the general formula I as definedabove, or an enantiomer, diastereomer, racemate, or pharmaceuticallyacceptable salt or solvate thereof.

In one particular embodiment, the method of the present invention is fortreatment of CILI.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Experimental Synthesis of 3-nitrooxymethyl-proxyl

A suspension of 3-carboxyproxyl (77.5 g, 0.416 mol) in drytetrahydrofuran (THF, 500 ml) was cooled to 0° C. Lithium aluminumhydride (2M solution in THF, 160 ml, 0.32 mol) was slowly added to thesuspension over 4 hours under nitrogen atmosphere. During the addition,the temperature was maintained below 5° C. After the completion ofaddition, the reaction mixture was stirred at 0-5° C. for 30 minutes andthen at room temperature for 3 hours. The reaction mixture was againcooled to 0° C. Sodium sulfate decahydrate solid (52 gm) was slowlyadded in small portions over 1 hour. The suspension was stirred furtherfor 1 hour. It was diluted with ethyl acetate (1 liter) and filtered.The cake was washed with ethyl acetate (500 ml) and the filtrate wasconcentrated on rotary evaporator. The residue obtained afterconcentration was suspended in 30% ethyl acetate-hexane (500 ml), andthe solid separated out was filtered and washed with 30% ethylacetate-hexane and dried under vacuum to give 3-hydroxymethyl-proxyl asyellow colored solid (56.320 g). The material was used as such for nextreaction. Concentrated nitric acid (90% solution, 100 ml) was cooled to0° C. using ice bath in 2 liter beaker. Concentrated sulfuric acid (100ml) was slowly added to nitric acid at 0° C. over 15 minutes. Themixture was stirred at 0-50° C. for 15 minutes. 3-Hydroxymethyl-proxyl(50 g, 0.29 mol) was added in small portions over 2 hours. After theaddition, the mixture was stirred at 0-5° C. for 1 hour, and quenchedwith crushed ice (˜1 kg). The mixture was then basified by slow additionof solid potassium carbonate (460 g) over 1 hour at 0° C. The basicmixture was stirred at room temperature for 30 minutes. The solidseparated out was filtered and washed with water (1 liter). The solidwas re-suspended in water (400 ml) and ethyl acetate (400 ml) added, andthe biphasic mixture was transferred into a separatory funnel. The ethylacetate layer was collected. Aqueous layer was extracted again withethyl acetate (400 ml). The combined organic extract was dried onanhydrous sodium sulfate and filtered. The filtrate was concentrated onrotary evaporator. The residue was dissolved ethyl acetate andtriturated with hexane. The solid separated out was collected byfiltration to produce 3-nitrooxymethyl-proxyl as pale yellow solid (39.7g). MS (ES⁺): m/z 218.3 (M+1).

Synthesis of (1-hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methylnitrate hydrochloride

A suspension of 3-nitrooxymethyl-proxyl (10.0 g, 0.046 mol) in ethanol(100 ml) was slowly treated with 4 M solution of hydrochloric acid indioxane (0.1 mol) at room temperature. The reaction mixture was thenstirred for 2 hours and concentrated on rotary evaporator. Dry ether wasadded and the solid separated out was filtered and dried under vacuum togive (1-hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl nitratehydrochloride as white solid (11.050 g). ¹H NMR (DMSO-d₆): δ 1.19-1.51(m, 12H), 2.02 (m, 1H), 2.48-2.52 (m, 1H), 3.45 (m, 1H), 4.56-4.66 (m,2H), 11.52 (bs, 1H), 11.86 (bs, 1H). MS (ES⁺): m/z 219.3 (M+1).

Example 1. Synthesis of[1-(acetyloxy)-2,2,5,5-tetramethylpyrrolidin-3-yl] methyl nitrate, 1a₁(R-107)

1-Hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl nitratehydrochloride (4.0 g, 0.0157 mol) was dissolved in THF (40 ml) andtreated with triethylamine (4.5 ml) and acetic anhydride (2 ml) at roomtemperature for 1 hour. The reaction mixture was concentrated anddissolved in ethyl acetate and water (50 ml each). Organic layer wascollected and dried over sodium sulfate. It was concentrated and theresidue was purified on silica gel column using 0-20% ethylacetate-hexane to give(1-acetoxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl nitrate as paleyellow oil (4.1 g). ¹H NMR (CDCl₃): δ 1.10 (s, 3H), 1.15 (s, 3H), 1.18(s, 3H), 1.21 (s, 3H), 1.59-1.65 (m, 1H), 1.88-1.93 (dd, J=7 and 12 Hz,1H), 2.11 (s, 3H), 2.37-2.41 (m, 1H), 4.36-4.51 (m 2H).

Example 2. Synthesis of[(1-propanoyloxy-2,2,5,5-tetramethylpyrrolidin-3-yl)] methyl nitrate,1a₂

1-Hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl nitratehydrochloride (1.0 g, 0.004 mol) was dissolved in THF (20 ml) andtreated with triethylamine (3 eq) and propionic anhydride (2 eq) at roomtemperature for 1 hour. The reaction mixture was concentrated anddissolved in ethyl acetate and water (50 ml each). The organic layer wascollected and dried over sodium sulfate. It was concentrated and theresidue was purified on silica gel column using 0-20% ethylacetate-hexane to give (1-propanoyloxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl nitrate as pale yellow oil (1.010 g). ¹H NMR(CDCl₃): δ 1.09 (s, 3H), 1.16 (s, 3H), 1.17 (s, 3H), 1.18-1.21 (m, 6H),1.58-1.65 (m, 1H), 1.85-1.92 (dd, J=7.5 and 12.9 Hz, 1H), 2.33-2.41 (m,2H), 2.37-2.41 (m, 1H), 4.34-4.50 (m 2H).

Example 3. Synthesis of [1-(2-methylpropanoyloxy-2,2,5,5-tetramethylpyrrolidin-3-yl)]methyl nitrate, 1a₃

1-Hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl nitratehydrochloride (1.04 g, 0.004 mol) was dissolved in THF (20 ml) andtreated with triethylamine (3 eq) and isobutyryl anhydride (1.5 eq) atroom temperature for 30 minutes. The reaction mixture was concentratedand dissolved in ethyl acetate and water (50 ml each). Organic layer wascollected and dried over sodium sulfate. It was concentrated and theresidue was purified on silica gel column using 25% ethyl acetate-hexaneto give[1-(2-methylpropanoyloxy-2,2,5,5-tetramethylpyrrolidin-3-yl)]methylnitrate as pale yellow oil (1.0 g). ¹H NMR (CDCl₃): δ 1.10-1.22 (m,18H), 1.61 (dd, J=12 Hz, 1H), 1.85-1.92 (dd, J=7.5 and 12 Hz, 1H),2.33-2.43 (m, 1H), 2.57-2.67 (m, 1H), 4.38-4.51 (m, 2H).

Example 4. R-107 is Converted Under Physiological Conditions to R-100

In an in vitro study,2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-yl acetate (R-107)was incubated in mouse or rat plasma at 37° C. at a concentration of 4mM, and the relative concentrations of this compound as well as of itscorresponding hydroxylamine,(1-hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl) methyl nitrate (R-105),and 3-nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy (R-100), weremeasured by HPLC. Table 4 shows the relative concentration of each oneof the compounds in mouse and rat plasma following 0.1, 1.5 and 20hours, indicating that R-107 is, in fact, a prodrug of its correspondinghydroxylamine compound upon hydrolysis of the ester bond (O—C(O)R₃ inthe general formula I), wherein the hydroxylamine compound is thenoxidized, in vivo, to its corresponding nitroxide derivative R-100.

TABLE 4 Relative concentrations of R-107, R-105 and R-100 uponincubation of R-107 in mouse or rat plasma Mouse Rat ConcentrationConcentration Time (hrs) R-100 R-105 R-107 R-100 R-105 R-107 0.1 0 0 3268 68 246 1.5 91 61 284 26 159 18 20 260 32 0 195 29 0

In the following two experiments, the pharmacokinetic of releasing R-100from R-107, via R-105 that is the reduced form of R-100, was studied invivo according to the protocol described herein.

Methods and Standards

100 μl aliquots plasma obtained from R-107-treated sheep or pig werethawed and 200 μl ice-cold ethanol was added to each 100 μl sample toprecipitate protein. Samples were then vortexed, placed on ice for 10minutes, and then spun down at room temperature for 15 minutes. A first100 μl supernatant from each spun sample was withdrawn and placed in alabeled HPLC micro vial to which 300 μl MPA (LC mobile phase A) wasadded, and the vial contents were then mixed and submitted for LCMSanalysis (kept at 4° C.). The concentration of R-107 was measured inthis sample as was the concentration of R105. A second 100 μlsupernatant from each spun sample was added to a labelled HPLC microvial to which 300 μl MPA and 2 μl of Reducing solution (ascorbic acid,1.0 mg/ml in water) were added. These vials were vortexed; incubated at50° C. for 1 hour with gentle shaking; and were then cooled to 4° C. andsubmitted for LCMS analysis. This sample was analyzed for R105, i.e.,reduced form of R-100, and the amount measured thus represents the sumof R-100 and R-105 originally present in the plasma. The concentrationof R-100 can be calculated from that of the R-105 in the second sampleminus that of R-105 in the first sample. However, the concentrations ofR-105 were seen to be low (approximately 10% or less of those of R-100)and so the results reported below as R-100 are not corrected for R-105.

Sets of standards (R-100, R-105 and R-107) were prepared by spikingindividually in pig plasma. 100 μl aliquots of these spiked plasmasamples, after thawing, were included in the above extraction protocol.

Analysis Methods

Analysis method were carried out with an Agilent HPLC stack, with columneluate directed, via a diverter valve, into the source of an AB Sciex‘Qstar Elite’ Hybrid TOF, high-resolution mass spectrometer. LC column:Phenomenex Kinetex C18 2.6 u 100 A, 2.1×50 mm. LC mobile phase A: H₂Owith 0.02% trifluoroacetic acid (TFA), 0.1% formic acid (HOF); Phase B:acetonitrile (ACN) with 0.02% TFA, 0.1% HOF. Mobile phase flow rate=500μl/min. Injection size: 8 μl, via Agilent 1100 u-WPS autosampler.

Tables 5 and 6 show the amount of R-107 and R-100 measured in the plasmaof a sheep (35 kg) at certain points in time following IV or IMadministration, respectively, of neat R-107 (0.8 ml, 880 mg, 25 mg/kg)over 10 minutes. For the purpose of comparison, Table 7 shows the amountof R-100 measured at certain points in time following IV administrationof R-100 in HPCD (20 mg/ml), 20 mg/kg bolus (the ratio between themolecular weights of R-107:R-100 is 269:217, so the dosing of 25 mg/kgand 20 mg/kg, R-107:R-100, is approximately equimolar).

TABLE 5 IV injection of neat R-107 to sheep Time post dose (hrs) R-100μg/ml R-107 μg/ml 0 0 0 0.1 12.04 5.5 0.4 8.87 1.21 0.5 7.10 0.2 1 3.140 2 1.25 not detected 4 0.43 not detected 6 0.18 not detected 8 0.12 notdetected 24 0.06 not detected

TABLE 6 IM injection of neat R-107 to sheep (no R-107 was detected) Timepost dose (hrs) R-100 μg/ml 0 — 0.25 0.14 0.5 0.2 1 0.3 2 0.32 4 0.38 80.33 24 0.17 48 not detected

TABLE 7 IV injection of neat R-100 to sheep (20 mg/kg bolus) Time postdose (hrs) R-100 μg/ml 0 0 0.08 6.72 0.17 6.11 0.25 5.31 0.5 2.72 1 1.632 0.7 4 0.28 8 0.03

Table 8 shows the amount of R-107 and R-100 measured in the plasma of apig (10 kg) at certain points in time following IV administration ofneat R-107 (0.8 ml, 880 mg, 88 mg/kg) over 6 minutes; Table 9 shows theamount of R-107 and R-100 measured in the plasma of a pig (10 kg) atcertain points in time following IV administration of R-107 diluted in 4parts of PEG400 (0.8 ml, 176 mg, 18 mg/kg) over 6 minutes; and Table 10shows the amount of R-107 and R-100 measured in the plasma of a pig (10kg) at certain points in time following oral administration of neatR-107 (0.8 ml, 880 mg, 88 mg/kg; given as a capsule per os).

TABLE 8 IV injection of neat R-107 to pig Time post dose (hrs) R-107μg/ml R-100 μg/ml −0.08 0 0 0.08 5.24 16.03 0.17 5.87 15.50 0.25 3.4316.63 0.5 1.32 16.76 1 0.36 13.63 2 0.26 7.36 6 not detected 0.70 24 notdetected 0.03

TABLE 9 IV injection of R-107 diluted in 4 parts of PEG400 to pig Timepost dose (hrs) R-107 μg/ml R-100 μg/ml −0.08 0 0 0.08 2.38 5.83 0.170.64 5.76 0.25 0.43 4.88 0.5 0.25 3.84 1 0.22 2.50 2 not detected 1.72 4not detected 0.78 8 not detected 0.20 24 not detected not detected

TABLE 10 Capsule (per os) of neat R-107 to pig (0.8 ml, 88 mg/kg) Timepost dose (hrs) R-107 ng/ml R-100 ng/ml −0.08 — — 0.08 1.319 0.964 0.170.715 1.504 0.5 0.345 1.424 1 0.11 1.257 2 0.222 0.609 3 0.11 1.607 40.122 2.031 8 not detected 0.387 24 not detected not detected

Example 5. R-107 is Effective in a Murine Model of Sepsis

In this study, Balb/c mice exposed to a lethal dose of E. colilipopolysaccharide (LPS; 10 mg/kg IP) are treated with R-107 (0, 20, 40,or 80 mg/kg/day, BID IP), with the initial dose given 1 hour after LPSinjection, and the mortality for the animal treated with LPS only (thecontrol group) vs. that for the animals treated with the various dosesof R-107 is compared.

In a satellite study, tissues and sera are collected at 16 hours, i.e.,15 hours after the first administration and 3 hours after the secondadministration of R-107, for analysis, and the level of creatinine,aspartate transminase (AST), alanine transaminase (ALT), bilirubin,amylase, lipase, and alkaline phosphatase (ALP), associated with kidney,pancreas, and liver functioning are measured.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 6. R-107 is Effective in a Sheep Model of Septic Pneumonia

In this study, a model of sheep suffering from Pseudomonas aeruginosa(PSA)-induced septic pneumonia and consequent pulmonary dysfunction isused. Female Merino sheep are operatively instrumented for chronic studywith Swan-Ganz®, i.e., pulmonary artery, and femoral artery catheters,and are randomly allocated to vehicle control or R-107 treatment group.Smoke inhalation injury (48 breaths of cotton smoke) is induced and2.4×10¹¹ colony-forming units (CFU) of PSA are instilled in the lung viabronchoscope under general anesthesia. The animal in the treatment groupare intravenously (IV) administered with a total of 80 mg/kg of R-107BID. Ringer's lactate solution is titrated IV to maintain hematocrit(hct) at baseline±3%. Measurements are taken at baseline and every 3hours during the 24 hours study period. Data are expressed as mean±SEM.Statistical analysis: two-way ANOVA and Bonferroni post hoc comparison.A p-value <0.05 is considered as statistically significant.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 7. The Effect of R-107 on MCT-Induced Changes in Systemic andPulmonary Arterial Pressure, and on MCT-Induced Pulmonary VascularRemodeling

In this study, adult male Sprague-Dawley rats (250-350 g) (3 groups) aretreated with a single subcutaneous injection of monocrotaline (MCT; 60mg/kg), a plant poison that induces a well-characterized experimentalmodel of pulmonary hypertension, or an equivalent volume of saline (2ml/kg; vehicle, control). After a period of 38 days in which ratsdevelop severe PAH, dosing with R-107 for 10 days is initiated, asfollows: group 1 (sham animals) does not receive MCT and is dosed withvehicle control in drinking water; group 2 is dosed with vehicle controlin drinking water; group 3 is dosed with R-107 (80-160 mg/kg/day, BIDIP). At the conclusion of the 10-day dosing period, rats areanesthetized and instrumented, and resting hemodynamic indices arerecorded.

At post-mortem, the lower lobe of the right lung is fixed with formalinsolution, and after paraffin embedding, 5 mm sections are stained withhaematoxylin and eosin and observed in a Dialux 22 Leitz (Wetziar,Germany) microscope. The score of lung fibrosis is assessed on sectionsstained with Masson Trichrome staining. For morphometric evaluations,all three lobes of right lung are inspected. For each lobe the vesselsof medium and small size that demonstrate edema and inflammatory cellsare counted. Results are expressed as the percentage of vesselspresenting indices of disease relative to the total number of vesselscounted in the sections. The percentage of vessels demonstratingthickening of the layer of smooth muscle in the tunica are alsoexpressed as a percentage relative to the total number of vesselscounted.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 8. R-107 is Effective in a Lamb Model of Pulmonary Hypertensionof the Newborn

In this study, a prospective, placebo-controlled, dose-escalation,randomized study is carried out in anesthetized,mechanically-ventilated, and muscle-relaxed newborn lambs whereinpulmonary hypertension of the newborn (PPHN) has been induced by inutero ligation of the ductus arteriosus. This clinically-relevantgold-standard model is characterized by severe PAH that is poorlyresponsive to inhaled nitric oxide (iNO), thus reflecting the 30-50% ofclinical PPHN non-responders to clinical iNO therapy. R-107 (0, 0.3,1.0, and 3.0 mg/kg/h IV; n=6 per group) is compared to iNO (40 ppm; n=6)over a 6 hour period of observation. Hemodynamic parameters aremonitored for response to treatment and post-treatment rebound pulmonaryarterial hypertension. The activity of R-107 is related to its plasmaconcentration, in order to construct a pharmacodynamic profile.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 9. R-107 is Effective in a Minipig Model of PeripheralHypertension

In order to test the efficacy of R-107 in peripheral hypertension (HTN),a series of 3 hemodynamic studies is carried out in conscious,instrumented, ambulatory, and telemetered minipigs (n=5) with HTNsecondary to an endovascular-created, unilateral, renovascular stenosis.Minipigs are treated with R-107 administered as an enterically-coatedoral capsule.

In Series A, the pharmacokinetic profile and hemodynamic effect of asingle administration of R-107 (3, 10, 20, 30 mg/kg) is correlated witha one-week washout period between successive dose levels. Animals aremonitored for 24 hours to collect continuous hemodynamic data andintermittent plasma concentrations of R-107 and its metabolites (0,0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 16, 20, and 24 hours). In Series B,the most promising 2 doses of R-107 from Series A are evaluated bycarrying out a repeat-dose study of R-107 administered every 6 hours(q6h) for a period of 3 days. The hemodynamic effects of R-107 arecorrelated with plasma peak and trough concentrations of R-107 and itsmetabolites obtained q6h. In Series C, the emergence of tachyphylaxis toR-107 in a 2 week repeat-dose study (q6h) is evaluated utilizing theoptimal dose determined in Series B, wherein hemodynamics are correlatedwith plasma pharmacokinetic determinations as in Series B.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 10. R-107 is Effective in a Rat Model of RenalIschemia-Reperfusion Injury

In this study, the effect of R-107 at the dose of 80 mg/kg is tested ina well characterized model of kidney ischemia and reperfusion in rats.Male Sprague-Dawley rats are placed onto a thermostatically-controlledheating mat, and body temperature is maintained at 38±10° C. by means ofa rectal probe attached to a homeothermic blanket. A tracheotomy isperformed to maintain airway patency and to facilitate spontaneousrespiration. A midline laparotomy is performed, and the bladder iscannulated. Both kidneys are located, and the renal pedicles, containingthe artery, vein, and nerve supplying each kidney, are carefullyisolated.

Rats (Groups 2 and 3) are allowed to stabilize for 30 minutes beforethey are subjected to bilateral renal occlusion for 30 minutes usingartery clips to clamp the renal pedicles. Reperfusion commenced once theartery clips are removed (control animals). Occlusion is verifiedvisually by change in the color of the kidneys to a paler shade andreperfusion by a blush. The other rats (Group 1), which underwentidentical surgical procedures similar to control animals but did notundergo bilateral renal clamping, are subjected to sham operation (shamoperated) and are maintained under anesthesia for the duration of theexperiment. At the end of all experiments, animals are killed by anoverdose of sodium thiopentone.

Upon completion of surgical procedures, the animals are randomlyallocated to Group 1: Sham (no ischemia); Group 2:(ischemia-reperfusion, I/R); and Group 3: (ischemia-reperfusion, I/R,and R-107 therapy). Urine is collected from the rats during thefollowing periods: (i) 2 hours prior to ischemia until 30 minutes priorto ischemia; (ii) from the onset of reperfusion until 1.5 hours afterthe onset of reperfusion; (iii) from 1.5 hours after the onset ofreperfusion until 3.0 hours after the onset of reperfusion; (iv) from 3hours after the onset of reperfusion until 4.5 hours after the onset ofreperfusion; and (v) from 4.5 hours after the onset of reperfusion until6.0 hours after the onset of reperfusion. Arterial blood samples areobtained at 0, 1.5 hours, 3.0 hours, 4.5 hours, and 6.0 hours after theonset of reperfusion. R-107 (80 mg/kg; Group 3) is administered as a 10minute IV infusion beginning 20 minutes after the onset of ischemia.

At the end of the reperfusion period, blood (1 ml) samples are collectedvia the carotid artery into S1/3 tubes containing serum gel, and thesamples are centrifuged (6000 rpm for 3 minutes) to separate plasma. Allplasma samples are analyzed for biochemical parameters within 24 hoursafter collection. Urine samples are collected during the reperfusionperiod, and the volume of urine produced is recorded. Urineconcentrations of Na⁺ are measured and are used in conjunction withplasma Na⁺ concentrations to calculate fractional excretion of sodium(FE_(Na)) using standard formulae, which is used as an indicator oftubular function. Plasma and urine concentrations of creatinine aremeasured as indicators of impaired glomerular function. Creatinineclearance is calculated using the formula UV/P, where U refers tocreatinine concentration in urine, V refers to urine volume/min and Prefers to serum creatinine. Plasma concentrations of neutrophilgelatinase-associated lipocalin (NGAL) are evaluated as indicated by thecommercial kit. Urine concentration of NGAL is evaluated as indicated bythe commercial kit.

Myeloperoxidase (MPO) activity in kidneys is used as an indicator ofpolymorphonuclear (PMN) cell infiltration using a method previouslydescribed. Briefly, at the end of the experiments, kidney tissue isweighed and homogenized in a solution containing 0.5% (wt/vol)hexadecyltrimethyl ammonium bromide dissolved in 10 mmol/1 potassiumphosphate buffer (pH 7.4) and centrifuged for 30 minutes at 20,000 g at4° C. An aliquot of supernatant is then removed and added to a reactionmixture containing 1.6 mmol/l tetramethylbenzidine and 0.1 mmol/lhydrogen peroxide (H₂O₂). The rate of change in absorbance is measuredspectrophotometrically at 650 nm. MPO activity is defined as thequantity of enzyme required to degrade 1 mmol of H₂O₂ at 37° C. and isexpressed in U/g wet tissue.

Levels of malondialdehyde (MDA) in kidneys are determined as anindicator of lipid peroxidation following a protocol describedpreviously. Briefly, kidney tissue is weighed and homogenized in a 1.15%(wt/vol) KCl solution. A 100 ml aliquot of homogenate is then removedand added to a reaction mixture containing 200 ml 8.1% (wt/vol) laurylsulfate, 1.5 ml 20% (vol/vol) acetic acid (pH 3.5), 1.5 ml 0.8% (wt/vol)thiobarbituric acid, and 700 ml distilled water. Samples are then boiledfor one hour at 95° C. and centrifuged at 3000×g for 10 minutes. Theabsorbance of the supernatant is measured spectrophotometrically at 650nm. MDA levels are expressed as μM/100 mg wet tissue.

At post-mortem, a 5 μm section of kidney is removed and placed informalin and processed through to wax. Five millimeter sections are cutand stained with hematoxylin and eosin. Histologic assessment of tubularnecrosis is determined semiquantitatively using a method modified fromMcWhinnie et al., Tissue Antigens, 1987, 29(4), 214-223. Random corticalfields are observed using a ×20 objective. A graticule grid (25 squares)is used to determine the number of line intersects involving tubularprofiles. One hundred intersections are examined for each kidney, and ascore from 0 to 3 is given for each tubular profile involving anintersection: 0=normal histology; 1=tubular cell swelling, brush borderloss, nuclear condensation, with up to one third of tubular profileshowing nuclear loss; 2=same as for score 1, but greater than one thirdand less than two thirds of tubular profile shows nuclear loss;3=greater than two thirds of tubular profile shows nuclear loss. Thetotal score for each kidney is calculated by addition of all 100 scores.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 11. R-107 is Effective in a Murine Model of RetinalIschemia-Reperfusion Injury

In this study, the efficacy of R-107 in subacute murine model of retinalischemia-reperfusion injury is tested, using male C57BL/6 mice (n=10 pergroup). Briefly, the anterior chamber of the right eye is cannulatedwith a 30 gauge needle attached to a line infusing sterile saline. Theintraocular pressure (IOP) is raised to 110 mmHg by elevating the salinereservoir. The left eye undergoes sham surgery, in which the needle isinserted into the anterior chamber without elevating IOP. R-107 isinjected (30 mg/kg/day IP BID) commencing directly before reperfusion. 2vehicle controls (normal saline and HPCD) is compared to drug therapy.Treatments are repeated twice daily thereafter until sacrifice. Theoutcome of these pharmacologic studies is quantitatively assessed byusing terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) to measure retinal cell death (after 3 days), confocal imagingto quantify survival of ganglion cell layer neurons (after 7 days), andelectroretinogram recordings to measure retinal neuronal function (after21 days).

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 12. R-107 is Effective in a Porcine Model of Acute RetinalIschemia-Reperfusion Injury

In this study, a porcine model of acute retinal ischemia-reperfusioninjury (n=6 per group) is induced by equilibration with an elevatedsaline reservoir to create a static IOP of 90 mmHg for 90 min. Task #1:Microvascular responsiveness: A dose-response of parenteral R-107 (0,10, 30, 80 mg/kg/day IV BID) is initiated 10 minutes before reperfusion.1 hour after reperfusion, retinal microvessels are harvested andcannulated for ex vivo analysis of vascular responsiveness toendothelium dependent and independent vasodilators. Task #2: Retinalfunction and morphology: R-107 is given 10 minutes before reperfusion(at a dose determined by Task #1) and its efficacy judged byelectroretinogram (1, 2, 7 days post-reperfusion) and retinal histology,ultrastructure, and drug concentration (Day 7).

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 13. R-107 is Effective in a Rat Model of MyocardialIschemia-Reperfusion Injury

In order to test the efficacy of R-107 in a myocardial ischemiareperfusion injury, male aged Sprague-Dawley rats are dosed with R-107(0, 10, 30, 80 mg/kg IV) 10 minutes prior to the end of a 30 minutesperiod of ischemia produced by occlusion of the left anterior descending(LAD) coronary artery. Cardiac function is monitored by a Millarcatheter for the determination of heart rate (HR), systolic anddiastolic blood pressure (BP), left ventricular (LV) systolic BP, LVend-diastolic BP (LVEDP), ±dP/dt, pressure-volume (PV) loop, and tau(τ). 3 hours post-ischemia, blood and myocardium are analyzed formorphologic, histochemical, and bioanalytical injury. The area ofnecrosis, as a % of area at risk (AAR), and the extent of the no-reflowzone are quantified.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 14. R-107 is Effective in a Minipig Model of LimbIschemia-Reperfusion Injury

In this study, the efficacy of R-107 in a limb ischemia reperfusioninjury (LIRI) is tested. In a large animal model of acute LIRI (n=6minipigs per group) induced by an endovascular balloon occlusion of thefemoral artery for 6 hours, parenteral R-107 (0, 10, 30, 80 mg/kg/dayTID) is initiated 10 minutes before reperfusion via direct arterialinfusion, followed by an IV infusion thereafter. At 7, 14, and 21 daysafter reperfusion, limb perfusion (by Doppler flow) is correlated withnerve conduction velocity (NCV, per electromyography, EMG), and musclestrength (by foot pressure on a walking surface) with simultaneousplasma concentrations of R-107.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 15. R-107 is Effective in a Rat Model of Contrast-InducedNephropathy

In order to test the efficacy of R-107 in contrast-induced nephropathy(CIN), a single-blinded study of CIN is carried out in Sprague-Dawleyrats (n=10 per group) subjected to dehydration, prostaglandin synthetaseinhibition, and an IV challenge of contrast media (CM). A sham injurygroup is compared to treatment of CM-challenged healthy rats with R-107(10, 20, 40 mg/kg/day in 5% dextrose, BID IV), N-acetylcysteine (NAC,100 mg/kg/day in 5% dextrose, BID), or vehicle control (hydroxypropylcyclodextrin, HPCD) initiated 10 min before CM administration andcontinued for 48 hours. Rats are evaluated at 48 hours for renalfunction (blood urea nitrogen to creatinine ratio, BUN:creatinine) andparameters reflecting renal injury, including protein release into theblood (NGAL; kidney injury molecule-1, KIM-1), pro-inflammatorytranscription factor expression (cytoplasmic IkBa degradation, nuclearp65 translocation), histologic damage (H&E), mitochondrial swelling(electron microscopy), and nuclear damage and DNA repair (PARPactivation and nitrosative stress as noted by poly(ADP-ribose) and3-nitrotyrosine (3-NT) tissue immunoreactivity).

In order to better model the patient population at greatest risk of CIN,the optimal dose of R-107 is then further evaluated, using the sameexperimental approach and design as before, but in Sprague-Dawley ratspreviously rendered diabetic 2 weeks earlier by injection ofstreptozotocin (STZ; 60 mg/kg IV). A control group of rats withSTZ-induced diabetes receive vehicle control. If efficacy of R-107 inthe diabetic group is less than in healthy rats, additional groups areevaluated at higher doses of R-107 in order to establish the optimalresponse in this clinically-relevant model of diabetes and CIN. Finally,we investigate whether further benefit may be obtained in the diabeticsetting by combining the administration of R-107 with aggressive volumeresuscitation, based on the apparent current clinical consensus thatincreased hydration ameliorates CIN. Diabetic rats in the elevatedhydration group receive 10 ml/kg IV normal saline (NS) bolusescoincident with the administration of R-107. A control group receives IVNS alone.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

Example 16. R-107 is Effective in a Murine Cl₂ Exposure Model

In this study, the effect of R-107 in treatment of CILI is tested.Balb/c mice are exposed in a cylindrical glass chamber that is flushedcontinuously for 60 minutes at a rate of 2 liters/minute with humidifiedgas obtained from a calibrated cylinder containing air and 400 ppm Cl₂.After the end of the 30 minute exposure, the chamber is opened and miceare removed and immediately placed in cages in room air. Two and sixhours after the conclusion of Cl₂ exposure, mice are administeredintraperitoneal (IP) with various doses of R-107. At 24 hourspost-exposure to the Cl₂-containing air, a midline incision from theneck to the pubis is created for access to the chest and abdominalcavities. Blood samples are obtained from the inferior vena cava justbefore sacrifice, the heart-lung block is rapidly excised, and thepulmonary circulation is flushed through the main pulmonary artery with20 ml of normal saline. The lungs are separated from the mediastinaltissues and are taken for biochemical assays and histologicalexamination (H&E staining). The following morphological criteria areused for scoring: grade 0, normal lung; grade 1, minimal edema orinfiltration of alveolar or bronchiolar walls; grade 3, moderate edemaand inflammatory cell infiltration without obvious damage to lungarchitecture; and grade 4, severe inflammatory cell infiltration withobvious damage to lung architecture. Similar studies may be conductedwith compounds of the general formula I other than R-107.

In a particular study, the effect of R-107 in treatment of CILI wastested according to the protocol above. Male Balc/c mice were divided tothe following 5 groups: (i) Sham (n=5); (ii) vehicle, HPCD (n=5); (iii)vehicle, PEG-400 (n=5); (iv) R-100 formulated in HPCD, 80 mg/kg/dose,total 160 mg/kg/day, IP (n=10); and (v) R-107 formulated in PEG-400, 100mg/kg/dose, total 200 mg/kg/day, IP (n=10). FIGS. 1A-1F showrepresentative photomicrographs demonstrating H&E-stained lung sectionstaken from Sham-operated mice (1A, 1B), animals treated with Cl₂+HPCD(1C), animals treated with Cl₂+saline (1D), animals treated withCl₂+R-100 (1E), and animals treated with Cl₂+R-107 (1F). FIG. 2 showsthat R-107 as well as its corresponding 1-pyrrolidinyloxy, R-100, whenadministered 2 and 6 hours post a 60 minute exposure to Cl₂ containingair, significantly attenuated CILI in mice 24 hours post exposure asexemplified by the improved histology scores.

In another study conducted according to the protocol described above,male Balc/c mice were divided to the following 5 groups: (i) Sham (n=2);(ii) vehicle, olive oil (n=4); (iii) R-107 formulated in olive oil, 10mg/kg/dose, total 20 mg/kg/day, IP (n=4); (iv) R-107 formulated in oil,30 mg/kg/dose, total 60 mg/kg/day, IP (n=4); and (v) R-107 formulated inoil, 80 mg/kg/dose, total 160 mg/kg/day, IP (n=4). Drug was administered2 and 6 hours post a 60 minute exposure to Cl₂ (400 ppm) containing air,and at 24 hours, the mice were sacrificed and lung histology examined.Table 11 shows that R-107 dose-responsively attenuated CILI in mice 24hours post exposure as exemplified by the improved histology scores.

TABLE 11 R-107 dose-responsively attenuates CILI in mice 24 hours postexposure as exemplified by the improved histology scores Histology scoreN^(o) mice 80 mg/kg 30 mg/kg 10 mg/kg vehicle Sham 1 2.2 2.8 3.0 3.8 0 21.5 2.2 3.0 4.8 0 3 2.0 2.4 3.5 3.4 4 1.8 3.2 3.2 4.7 Mean 1.88 2.643.18 4.17 0.00 SD 0.2986 0.4488 0.2375 0.6611 0 SE 0.1493 0.2244 0.11870.3306 0

Example 17. R-107 is Effective in a Rat Model of Erectile Dysfunction

In order to test the efficacy of R-107 in erectile dysfunction (ED), aprospective, placebo-controlled, randomized, single-blinded study of EDis carried out in anesthetized Sprague-Dawley rats (n=10 per group). Asham injury group is compared to treatment with R-107 (80 mg/kg/day BIDIV) initiated 10 minutes before crush injury of the cavernosal nerves ofthe penis and continued for 1 week. After 1 week, rats undergo in vivoneurogenic-mediated evaluation of the erectile response. At thecompletion of the physiologic evaluation, rats are euthanized andtissues removed for biochemical, morphologic, and inflammatory analyses.The crush injury is produced as follows: After IP injection of sodiumpentobarbital (50 mg/kg) for anesthesia, the abdomen is shaved andprepared with an iodine-based solution and a lower midline abdominalincision is made. The prostate gland is exposed and cavernosal nervestracking posterolateral are identified and isolated. In a sham groupthere is no further surgical manipulation. In the remaining groups crushinjury is created using a hemostat clamp for 3 minutes. The abdomen isthen closed in 2 layers in all rats. 1 week after surgical intervention,all rats undergo an in vivo neurogenic-mediated erectile response study.Rats are anesthetized with pentobarbital (50 mg/kg IP) and placed on athermally regulated surgical table. The trachea is cannulated usingPE-240 polyethylene tubing to maintain a patent airway and the animalsbreath room air enriched with 95% O₂/5% CO₂. A carotid artery iscannulated with PE-50 tubing to continuously measure systemic bloodpressure (MAP) using a Statham transducer attached to a data acquisitionsystem and connected to a computer. A 25 gauge needle filled withheparin (250 U/ml) and connected to PE-50 tubing is inserted into theright crura and connected to a pressure transducer to permit continuousmeasurement of intracavernous pressure (ICP). The cavernosal nerve (CN)is identified posterolateral to the prostate on 1 side and an electricalstimulator with a stainless steel bipolar hook is placed around the CN.The CN is stimulated with a square pulse stimulator (Grass Instruments,Quincy, Mass.). CN stimulation (CNS) at a frequency of 15 Hz and pulsewidth of 30 sec is performed in each rat. CNS at 2.5, 5, and 7.5 V isused in the current protocol to achieve significant consistent erectileresponses. The duration of stimulation is 1 minute with a rest period of3-5 minutes between subsequent CNSs. The total erectile response ortotal ICP is determined by the area under the curve in mm Hg per secondfrom the beginning of CNS until ICP returned to baseline orprestimulation pressure. The ICP/MAP ratio at the peak erectile responseis determined to control for variations in MAP.

Similar studies may be conducted with compounds of the general formula Iother than R-107.

The invention claimed is:
 1. A method for reducing oxidative stress orendothelial dysfunction in a mammal in need thereof, said mammalsuffering from a wound; diabetic retinopathy; diabetic nephropathy; ahemolytic disease selected from the group consisting of spherocytosis,glucose-6-phosphate dehydrogenase deficiency, sickle cell disease,thalassemia, and paroxysmal nocturnal hemoglobinuria; a cardiovasculardisease selected from the group consisting of ischemic heart disease,angina pectoris, myocardial ischemia reperfusion injury and infarction,acute and chronic limb ischemia reperfusion injury, congestive heartfailure, atherosclerosis, peripheral arterial hypertension cardiacarrhythmias, idiopathic pulmonary hypertension, pulmonary hypertensionassociated with idiopathic pulmonary fibrosis, pulmonary hypertensionassociated with hemolytic disease, primary pulmonary hypertension of thenewborn, and pulmonary hypertension secondary to congenitaldiaphragmatic hernia and meconium aspiration; a neurodegenerative orneurological disorder selected from the group consisting of Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, multiplesclerosis, convulsive (seizure) disorders, acquired immune deficiencysyndrome-dementia and a disorder which involves learning and memoryprocesses; or vaginal dryness, said method comprising administering tosaid mammal an effective amount of a compound of formula I:

or an enantiomer, diastereomer, racemate, or a pharmaceuticallyacceptable salt thereof, wherein each R₁ is independently selected fromthe group consisting of H, —OH, —COR₄, —COOR₄, —OCOOR₄, —OCON(R₄)₂,—(C₁-C₁₆)alkylene-COOR₄, —CN, —NO₂, —SH, —SR₄, —(C₁-C₁₆)alkyl,—O—(C₁-C₁₆)alkyl, —N(R₄)₂, —CON(R₄)₂, —SO₂R₄, —SO₂NHR₄, —S(═O)R₄, and anitric oxide donor group of the formula —X₁—X₂—X₃, wherein X₁ is absentor selected from the group consisting of —O—, —S— and —NH—; X₂ is absentor is (C₁-C₂₀)alkylene optionally substituted by one or more —ONO₂groups; and X₃ is —NO or —ONO₂, provided that at least one R₁ group is anitric oxide donor group; each R₂ is independently selected from thegroup consisting of (C₁-C₁₆)alkyl, (C₂-C₁₆)alkenyl, and (C₂-C₁₆)alkynyl;R₃ is selected from the group consisting of (C₁-C₁₀)alkyl,(C₃-C₁₀)cycloalkyl, (C₆-C₁₄)aryl, and 4-12-membered heterocyclyl, eachof which may optionally be substituted with —OH, —COR₅, —COOR₅,—(C₁-C₈)alkylene-COOR₅, —CN, —NO₂, —(C₁-C₈)alkyl, —O—(C₁-C₈)alkyl,—N(R₅)₂, —CON(R₅)₂, —SO₂R₅, —SO₂NHR₅, or —S(═O)R₅; each R₄ isindependently selected from the group consisting of H, (C₁-C₈)alkyl,(C₃-C₁₀)cycloalkyl, (C₆-C₁₄)aryl, and 4-12-membered heterocyclyl, eachof which other than H may optionally be substituted with —OH, —COR₅,—COOR₅, —OCOOR₅, —OCON(R₅)₂, —(C₁-C₈)alkylene-COOR₅, —CN, —NO₂, —SH,—SR₅, —(C₁-C₈)alkyl, —O—(C₁-C₈)alkyl, —N(R₅)₂, —CON(R₅)₂, —SO₂R₅,—SO₂NHR₅, or —S(═O)R₅; each R₅ is independently selected from the groupconsisting of H, (C₁-C₈)alkyl, (C₃-C₁₀)cycloalkyl, (C₆-C₁₄)aryl, and4-12-membered heterocyclyl; and n is 1, 2 or
 3. 2. The method of claim1, wherein each R₁ is independently H, —COOR₄, —CON(R₄)₂, or a nitricoxide donor group of the formula —X₁—X₂—X₃; and R₄ is H.
 3. The methodof claim 1, wherein each R₂ is independently (C₁-C₈)alkyl.
 4. The methodof claim 3, wherein each R₂ is identical.
 5. The method of claim 1,wherein R₃ is (C₁-C₈)alkyll, (C₁-C₈)alkylene-OH, (C₁-C₈)alkylene-N(R₅)₂,or (C₁-C₈)alkylene-COOR₅, wherein each R₅ is independently H, or(C₁-C₈)alkyl.
 6. The method of claim 1, wherein X₁ is absent or —O—. 7.The method of claim 1, wherein: each R₁ is independently H, —COOR₄,—CON(R₄)₂, or a nitric oxide donor group of the formula —X₁—X₂—X₃; eachR₂ is independently (C₁-C₈)alkyl; R₃ is (C₁-C₈)alkyl,(C₁-C₈)alkylene-OH, (C₁-C₈)alkylene-N(R₅)₂, or (C₁-C₈)alkylene-COOR₅;each R₄ is independently H; each R₅ is independently H or (C₁-C₈)alkyl;and X₁ is absent or —O—.
 8. The method of claim 1, wherein: (i) n is 1;and one or two of the carbon atoms at positions 3 or 4 of thepyrrolidine ring are linked to a nitric oxide donor group of the formula—X₁—X₂—X₃; (ii) n is 2; and one or more of the carbon atoms at positions3, 4 or 5 of the piperidine ring are linked to a nitric oxide donorgroup of the formula —X₁—X₂—X₃; or (iii) n is 3; and one or more of thecarbon atoms at positions 3, 4, 5 or 6 of the azepane ring are linked toa nitric oxide donor group of the formula —X₁—X₂—X₃.
 9. The method ofclaim 8, wherein said compound has two, three or four identical ordifferent nitric oxide donor groups of the formula —X₁—X₂—X₃.
 10. Themethod of claim 8, wherein each nitric oxide donor group of the formula—X₁—X₂—X₃ is independently —ONO₂, —(C₁-C₆)alkylene-ONO₂, or—O—(C₁-C₆)alkylene-ONO₂, wherein said alkylene is optionally substitutedby one or more —ONO₂ groups.
 11. The method of claim 10, wherein n is 1;each R₂ is independently methyl; R₃ is methyl, ethyl, or isopropyl; and(i) R₁ linked to the carbon atom at position 3 of the pyrrolidine ringis —CH₂—ONO₂; and R₁ linked to the carbon atom at position 4 of thepyrrolidine ring is H; (ii) R₁ linked to the carbon atom at position 3of the pyrrolidine ring is —ONO₂; and R₁ linked to the carbon atom atposition 4 of the pyrrolidine ring is H; (iii) each R₁ linked to thecarbon atoms at positions 3 and 4 of the pyrrolidine ring isindependently —CH₂—ONO₂; or (iv) each R₁ linked to the carbon atoms atpositions 3 and 4 of the pyrrolidine ring is independently —ONO₂. 12.The method of claim 11, wherein said compound is selected from the groupconsisting of: 2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-ylacetate; 2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-ylpropionate; and 2,2,5,5-tetramethyl-3-(nitrooxymethyl)pyrrolidin-1-ylisobutyrate, or an enantiomer, diastereomer, racemate, or apharmaceutically acceptable salt thereof.
 13. The method of claim 10,wherein n is 2; each R₂ is independently methyl; R₃ is methyl, ethyl, orisopropyl; and (i) R₁ linked to the carbon atom at position 3 of thepiperidine ring is —CH₂—ONO₂; and each R₁ linked to the carbon atoms atpositions 4 and 5 of the piperidine ring is independently H; (ii) R₁linked to the carbon atom at position 3 of the piperidine ring is —ONO₂;and each R₁ linked to the carbon atoms at positions 4 and 5 of thepiperidine ring is independently H; (iii) R₁ linked to the carbon atomat position 4 of the piperidine ring is —CH₂—ONO₂; and each R₁ linked tothe carbon atoms at positions 3 and 5 of the piperidine ring isindependently H; (iv) R₁ linked to the carbon atom at position 4 of thepiperidine ring is —ONO₂; and each R₁ linked to the carbon atoms atpositions 3 and 5 of the piperidine ring is independently H; (v) each R₁linked to the carbon atoms at positions 3 and 4 of the piperidine ringis independently —CH₂—ONO₂; and R₁ linked to the carbon atom at position5 of the piperidine ring is H; (vi) each R₁ linked to the carbon atomsat positions 3 and 4 of the piperidine ring is independently —ONO₂; andR₁ linked to the carbon atom at position 5 of the piperidine ring is H;(vii) each R₁ linked to the carbon atoms at positions 3 and 5 of thepiperidine ring is independently —CH₂—ONO₂; and R₁ linked to the carbonatom at position 4 of the piperidine ring is H; (viii) each R₁ linked tothe carbon atoms at positions 3 and 5 of the piperidine ring isindependently —ONO₂; and R₁ linked to the carbon atom at position 4 ofthe piperidine ring is H; (ix) each R₁ linked to the carbon atoms atpositions 3, 4 and 5 of the piperidine ring is independently —CH₂—ONO₂;or (x) each R₁ linked to the carbon atoms at positions 3, 4 and 5 of thepiperidine ring is independently —ONO₂.
 14. The method of claim 10,wherein n is 3; each R₂ is independently methyl; R₃ is methyl, ethyl, orisopropyl; and (i) R₁ linked to the carbon atom at position 3 of theazepane ring is —CH₂—ONO₂; and each R₁ linked to the carbon atoms atpositions 4, 5 and 6 of the azepane ring is independently H; (ii) R₁linked to the carbon atom at position 3 of the azepane ring is —ONO₂;and each R₁ linked to the carbon atoms at positions 4, 5 and 6 of theazepane ring is independently H; (iii) R₁ linked to the carbon atom atposition 4 of the azepane ring is —CH₂—ONO₂; and each R₁ linked to thecarbon atoms at position 3, 5 and 6 of the azepane ring is independentlyH; (iv) R₁ linked to the carbon atom at position 4 of the azepane ringis —ONO₂; and each R₁ linked to the carbon atoms at position 3, 5 and 6of the azepane ring is independently H; (v) each R₁ linked to the carbonatoms at positions 3 and 4 of the azepane ring is independently—CH₂—ONO₂; and each R₁ linked to the carbon atoms at positions 5 and 6of the azepane ring is independently H; (vi) each R₁ linked to thecarbon atoms at positions 3 and 4 of the azepane ring is independently—ONO₂; and each R₁ linked to the carbon atoms at positions 5 and 6 ofthe azepane ring is independently H; (vii) each R₁ linked to the carbonatoms at positions 3 and 5 of the azepane ring is independently—CH₂—ONO₂; and each R₁ linked to the carbon atoms at positions 4 and 6of the azepane ring is independently H; (viii) each R₁ linked to thecarbon atoms at positions 3 and 5 of the azepane ring is independently—ONO₂; and each R₁ linked to the carbon atoms at positions 4 and 6 ofthe azepane ring is independently H; (ix) each R₁ linked to the carbonatoms at positions 3 and 6 of the azepane ring is independently—CH₂—ONO₂; and each R₁ linked to the carbon atoms at positions 4 and 5of the azepane ring is independently H; (x) each R₁ linked to the carbonatoms at positions 3 and 6 of the azepane ring is independently —ONO₂;and each R₁ linked to the carbon atoms at positions 4 and 5 of theazepane ring is independently H; (xi) each R₁ linked to the carbon atomsat positions 3, 4 and 5 of the azepane ring is independently —CH₂—ONO₂;and R₁ linked to the carbon atom at position 6 of the azepane ring is H;(xii) each R₁ linked to the carbon atoms at positions 3, 4 and 5 of theazepane ring is independently —ONO₂; and R₁ linked to the carbon atom atposition 6 of the azepane ring is H; (xiii) each R₁ linked to the carbonatoms at positions 3, 4 and 6 of the azepane ring is independently—CH₂—ONO₂; and R₁ linked to the carbon atom at position 5 of the azepanering is H; (xiv) each R₁ linked to the carbon atoms at positions 3, 4and 6 of the azepane ring is independently —ONO₂; and R₁ linked to thecarbon atom at position 5 of the azepane ring is H; (xv) each R₁ linkedto the carbon atoms at positions 3, 4, 5 and 6 of the azepane ring isindependently —CH₂—ONO₂; or (xvi) each R₁ linked to the carbon atoms atpositions 3, 4, 5 and 6 of the azepane ring is independently —ONO₂. 15.The method of claim 10, wherein n is 1; each R₂ is independently methyl;R₃ is methyl, ethyl, or isopropyl; and (i) R₁ linked to the carbon atomat position 3 of the pyrrolidine ring is —CH₂—ONO₂; and R₁ linked to thecarbon atom at position 4 of the pyrrolidine ring is —CONH₂; or (ii) R₁linked to the carbon atom at position 3 of the pyrrolidine ring is—ONO₂; and R₁ linked to the carbon atom at position 4 of the pyrrolidinering is —CONH₂.
 16. The method of claim 10, wherein n is 2; each R₂ isindependently methyl; R₃ is methyl, ethyl, or isopropyl; and (i) R₁linked to the carbon atom at position 3 of the piperidine ring is—CH₂—ONO₂; R₁ linked to the carbon atom at position 4 of the piperidinering is —COOH; and R₁ linked to the carbon atoms at position 5 of thepiperidine ring is H; or (ii) R₁ linked to the carbon atom at position 3of the piperidine ring is —ONO₂; R₁ linked to the carbon atom atposition 4 of the piperidine ring is —COOH; and R₁ linked to the carbonatoms at position 5 of the piperidine ring is H.
 17. The method of claim10, wherein: n is 2; each R₂ is independently methyl; R₃ is methyl,ethyl, or isopropyl; R₁ linked to the carbon atom at position 3 of thepiperidine ring is H; R₁ linked to the carbon atom at position 4 of thepiperidine ring is —O—CH₂—CH(ONO₂)CH₂—ONO₂; and R₁ linked to the carbonatoms at position 5 of the piperidine ring is H.
 18. The method of claim1, wherein said mammal suffers from a wound.
 19. The method of claim 18,wherein said mammal suffers from a diabetic wound.
 20. The method ofclaim 1, wherein said mammal is a human.